BRPI0509847B1 - p-selectin binding antibody, its method of preparation and use, vector, composition and kit - Google Patents

Abstract

anti-p-selectin antibodies. The present invention relates to anti-β selectin antibodies, and in particular anti-β selectin antibodies and variants thereof which contain a human derived derivative fc and do not bind complement factor c1q. These antibodies have novel and inventive properties, causing a benefit to a patient suffering from critical limb ischemia or occlusive peripheral artery disease (cli / paod).

Description

Descriptive Report of the Invention Patent for ANTIBODY THAT CONNECTS TO P-SELETINE, ITS PREPARATION AND USE METHOD, VECTOR, COMPOSITION AND KIT.

The present invention relates, in general, to anti-P-selectin antibodies and, in particular, to anti-P-selectin antibodies that do not bind to complement factor C1q. Preferably, these antibodies are human or humanized antibodies.

P-selectin (CD62P, GMP-140, PADGEM, LECAM-3) is a 140 kDa calcium-dependent carbohydrate-binding protein that is expressed on the surfaces of activated platelets and endothelium in response to thrombin and other agonists (McEver and others, J Biol Chem 270: 11025 (1995); Varki, Proc Natl Acad Sci USA 91: 7390 (1994); Springer TA, Annu Rev Physiol 57: 827 (1995)). In both cell types, P-selectin is stored in secretory granules, that is, z-granules in platelets and WeibelPalade bodies in endothelial cells (McEver et al., J Clin Invest 84:92 (1984)). It is a type I transmembrane glycoprotein which is composed of an NH2-terminal lecithin domain, followed by an EGF-like domain, nine short consensus repetitions with homology to complementary regulatory proteins, a transmembrane domain and a short cytoplasmic tail ( Johnston et al., Cell 56: 1033 (1989)). The structure of P-selectin is similar to two other members of the selectin family, E- and L-selectin, which are expressed on cytokine-activated endothelial cells (E-selectin) or constitutively expressed on most leukocyte classes (L -selectin).

All selectins are known to bind with low affinity to small sialylated, fucosylated oligosaccharides, such as sially Lewis x (sLe ^x ; Foxall et al., J Cell Biol 117: 895 (1992); Varki, Curr Opin Cell Biol 257: 257 (1992)). P- and L-selectin, but not E-selectin, also bind to sulfated carbohydrates in particular, such as heparin sulfate (for review, see McEver and Cummings, J Clin Invest 100: S97 (1997)). Ligands with high affinity for P-selectin are mucin-like glycoproteins (McEver et al., J Biol Chem 270: 11025 (1995)), which consist of a polypeptide backbone with clusters of O-glycans

Petition 870190002756, of 10/01/2019, p. 7/16 stalylated. A sialomucin ligand to which P-selectin binds is preferably P-selectin glycoprotein-1 ligand (PSGL-1, CD162), which is normally expressed as a homodimer with two disulfide-linked subunits. relative molecular masses of approximately 5 120 kDa by circulating leukocytes. The P-selectin binding site is located at the extreme NhVterminal part of PSGL-1. Through its binding to its ligand, P-selectin mediates the roll of leukocytes on platelets and activated endothelial cells. The rolling process effectively reduces the speed of the leukocyte movement, which is a prerequisite 10 for firm adhesion and subsequent leukocyte transmigration to the subendothelium, but also for the accumulation of leukocytes in thrombi.

Studies using P-selectin-deficient mice and P-selectin-specific blocking antibodies have shown that P-selectin participates in the pathophysiology of numerous acute and chronic inflammatory diseases15, including ischemia / reperfusion injury (Winn et al., J Clin Invest 92 : 2042 (1993); Massberg et al., Blood 92: 507 (1998)). In addition, there is a clear contribution of P-selectin to cardiovascular diseases that have an inflammatory component, such as atherosclerosis (Collins et al., J Exp Med 191: 189 (2000); Johnson et al., J Clin Invest 99: 1037 (1997 )), 20 restenosis (Manka et al., Circulation 103: 1000 (2001); Bienvenu et al., Circulation 103: 1128 (2001)) and thrombosis (Kumar et al., Circulation 99: 1363 (1999); Andre et al., Proc Natl Acad Sei USA 97: 13835 (2000); Blann et al., Br. J. Haematol 108: 191 (2000); Myers et al., Thromb Haemostasis 85: 423 (2001). Evidently, the inhibition of P25 selectin function would be effective as a therapy in various diseases involving leukocyte adherence to the vascular endothelium or platelets (see, for example, WO 93/06863).

Antibodies against P-selectin have been described in the prior art and investigated for their anti-inflammatory and anti-thrombotic effects. US Patent 4,783,399 and WO 93/06863 describe mouse monoclonal antibodies against P-selectin reactive with activated platelets. Geng JG et al. (J. Biol. Chem., 266 (1991) 22313-22318) des3 create mouse monoclonal antibodies that bind to the amino acid (aa) fragment of P-selectin aa 60-75 (Cys to Glu, counted according to the P16109 sequence in Swiss-Prot, which includes the signal sequence WO 93/21956 refers to mouse monoclonal antibodies against P-selectin and humanized antibodies of the lgG1 subclass that compete with a defined antibody, binding in the presence of the aa 60-75 fragment of P-selectin) and in the absence of calcium ions.

None of the mouse monoclonal antibodies mentioned against human P-selectin is useful for the treatment of human patients. A humanized antibody against P-selectin of the human lgG1 subclass mentioned in WO 93/21956 is in preclinical development (www.rnrctechnoloqv.org) · SUMMARY OF THE INVENTION

The invention relates to antibodies characterized by the fact that said antibodies bind to P-selectin and do not bind to complement factor C1q. Preferably, the antibodies do not bind to the human Fcy receptor on NK cells. The antibodies according to the invention contain an Fc part derived from human origin. Preferably, these antibodies are human or humanized antibodies. Antibodies have new and inventive properties that bring a benefit to a patient who is suffering from inflammatory and thrombotic disorders, especially from peripheral artery occlusive disease (PAOD) and critical limb ischemia (CLI).

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows that the antibodies of the invention inhibit the adhesion of leukocyte-like HL60 cells to the purified P-selectin coated on microtiter slides. The mutated antibodies are more potent than the original antibody without mutation.

Figure 2 shows the inhibitory activity of the antibodies of the invention in the rosetting assay that measures the adhesion of activated platelets to HL60 cells.

Figures 3a and 3b represent the cross-reactivity of the antibodies of the invention with rat P-selectin and cynomologus. Figure 3a: Anti-P-selectin antibodies do not affect the adhesion of thrombin-activated rat platelets to HL60 cells, whereas the commercially available polyclonal anti-Pselectin antibody (Pharmingen 09361 A) inhibits this interaction. Figure 3b: The antibodies of the invention inhibit the adhesion of activated cynomologus platelets to HL60 cells.

Figures 4a-c demonstrate the selectivity of antibodies to P-selectin vs. E- and L-selectin through representative binding curves on P-, E- and L-selectin transfectants. The a10 antibodies in accordance with the invention bind to P-selectin / CHO cells with EC ₅₀ values in the range of 0.01 and 0.07 pg / ml. EC values of ₅ o on Eselectin / CHO cells and L-selectin / 300.19 cells are preferably above 100 μg / ml.

Figure 5 represents the inhibitory activity of antibodies from the invention in a fully human flow system. They inhibit the adhesion of human leukocytes to the platelet monolayer in a concentration-dependent manner at a shear rate of 65 / s.

Figure 6 represents the inhibitory effect of the antibodies of the invention on leukocyte adhesion to human endothelial cells expressing P-selectin. Figure 6a shows the total inhibition of leukocyte adhesion in% of the control. Figure 6b shows, representatively, the inhibitory effect of one of the antibodies on the absolute number of the different leukocyte subsets.

DETAILED DESCRIPTION OF THE INVENTION

I, Definitions

The term P-selectin refers to a 140 kDa protein expressed by human platelets and endothelial cells, as described by Hsu-Lin et al., J Biol Chem 259: 9121 (1984) and Mc Ever et al, J Clin Invest 84: 92 (1989). This type of transmembrane glycoprotein comprises 30 of an NH2-terminal lecithin domain, followed by a domain similar to epidermal growth factor (EGF) and nine repeated consensus domains. It is anchored in the membrane through a single transmembrane / y domain and contains a small cytoplasmic tail. The present invention provides antibodies which are capable of inhibiting one or more of the biological activities mediated by P-selectin, for example, its inflammatory or thrombotic activity. The antibodies bind to P-selectin and act through interference with the binding of P-selectin to its ligand.

The term P-selectin ligand preferably refers to the high affinity and biologically relevant ligand of P-selectin, such as the mucin-like glycoprotein P-selectin-1 glycoprotein-1 (PSGL-1), as described by Moore and others; J Cell Biol 118: 2445 (1992), Sako et al., Cell 75: 1179 (1993). PSGL-1 is a type 1 membrane protein with an extracellular domain rich in serines, threonines and prolines, including a series of decameric repeats linked with clusters of sialylated O-glycans. It is normally expressed as a homodimer with two disulfide-linked subunits with relative molecule masses of approximately 120 kDa by circulating leukocytes. The P-selectin binding site is located on the extreme NExtermini part of PSGL-1. Recently, sialomucin GPIba, which is expressed by platelets and has structural similarities to PSGL-1, has been shown to be a platelet ligand for P-selectin (Romo et al., J Exp Med 190: 803 (1999). The physiological consequences of binding GPIba to P-selectin are still under investigation, the interaction, however, being likely to contribute to platelet rolling and adherence to activated endothelial cells (Berndt et al., Thromb Haemost 86: 178 (2001). P-selectin also binds with low affinity to small sialylated, fucosylated oligosaccharides, such as sialyl Lewis x (Foxall et al., J Cell Biol 117: 895 (1992), Varki, CurrOpin Cell Biol 257 (1992) and sulfated carbohydrates in particular , such as heparin sulfate (McEver et al., J Biol Chem 270: 11025 (1995).

The term antibody encompasses the various forms of antibodies, preferably monoclonal antibodies including, but not limited to, whole antibodies, antibody fragments, human antibodies, humanized antibodies, chimeric antibodies and genetically engineered antibodies (variant or mutant antibodies), as insofar as the characteristic properties according to the invention are retained. Especially preferred are human or humanized monoclonal antibodies, especially as recombinant human antibodies.

The terms monoclonal antibody or monoclonal antibody composition, as used herein, refer to the preparation of antibody molecules from a composition with a single amino acid.

The term chimeric antibody refers to a monoclonal antibody comprising a variable region, that is, a binding region, of a source or species and at least part of a constant region derived from a different source or species, usually prepared using Recombinant DNA. Chimeric antibodies comprising a murine variable region and a human constant region are especially preferred. Such murine / human chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments that encode variable regions of murine immunoglobulin and DNA segments that encode constant regions of human immunoglobulin. Other forms of chimeric antibodies covered by the present invention are those in which the constant region has been modified or altered with respect to that of the original antibody to generate the properties according to the present invention, especially with regard to C1q binding and / or binding to the Fc (FcR) receptor. Such chimeric antibodies are also referred to as class-switched antibodies. Methods for producing chimeric antibodies involve conventional methods of recombinant DNA and gene transfection now well known in the art. See, for example, Morrison, S.L. and others, Proc. Natl. Acad. Know. USA 81 (1984) 68516855; US Patent Nos. 5,202,238 and 5,204,244.

The term humanized antibody refers to antibodies in which the network or regions of complementarity determination (CDR) have been modified to comprise the CDR of an immunoglobulin of different specificity when compared to that of the original immunoglobulin. In a preferred embodiment, a murine CDR is grafted into the network region of a human antibody to prepare the humanized antibody. See, for example, Riechmann, L. et al., Nature 332 (1988) 323-327; and Neuberger, M.S. et al., Nature 314 (1985) 268-270. Particularly preferred CDRs correspond to those representing sequences that recognize the aforementioned antigens for chimeric and bifunctional antibodies. Other forms of "humanized antibodies covered by the present invention are those in which the constant region has been modified or altered with respect to that of the orifinal antibody to generate the properties according to the invention, especially with regard to C1q binding and / or binding to the Fc (FcR) receptor.

The term human antibody, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well known in the art (van Dijk and van de Winkel, Curr Opin Pharmacol 5: 368 (2001). Human antibodies can also be produced in transgenic animals (for example, mice) that are capable, when immunized, to produce a total repertoire of human antibodies in the absence of endogenous immunoglobulin production. The transfer of a human germline immunoglobulin gene set to such germline mutant mice will result in the production of human antibodies when stimulated with antigen (see, for example, Jakobovits and others, Proc. Natl. Acad. Sci. USA, 90: 2551-2555 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggemann et al., Year emjmmuno., 7:33 (1993) )). Human antibodies can also be produced in phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1992); Marks et al., J. Mol. Biol, 222: 581 (1991)) The techniques of Cole et al and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, page 77 (1985) and Boerner et al., J. Immunol., 147 (1): 86-95 ( 1991)). As already mentioned for chimeric and humanized antibodies according to the invention, the term human antibody, as used here, also comprises such antibodies which are modified in the constant region to generate the properties according to the invention, especially with respect to binding to the C1q and / or connection to the FcR. In addition, the invention comprises human antibodies which bind to C1q and / or FcR. Such human antibodies are characterized by high selectivity for P-selectin vs. E- and L-selectin. Such antibodies according to the invention bind to cells that express Pselectin with EC ₅ o values in the range of 0.01 and 0.07 pg / ml. EC50 values on cells expressing E-selectin and L-selectin are preferably above 100 pg / ml. Such antibodies are preferably useful as intermediates for the manufacture of human antibodies with the properties according to the invention.

The term recombinant human antibody, as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, all antibodies isolated from a host cell, such as an NS0 or CHO cell, or from an animal (for example, a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected in a host cell. Such recombinant antibodies have variable and constant regions in a structured way. Recombinant human antibodies according to the invention were subjected to somatic hypermutation in vivo. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although derived and related to the human germline VH and VL sequences, may not naturally exist within the germline repertoire of human antibody in vivo.

The variable region (variable region of the light chain (VL), variable region of the heavy chain (VH)), as used here, denotes each of the pairs of light and heavy chains which are directly involved in the binding of the antibody to the antigen. The domains of human light and heavy chains have the same general structure and each domain comprises four network regions (RR) whose sequences are widely conserved, connected by three hypervariable regions (or regions of determination of end ⁹ .. ·. ·.: * · · * · ·?: «· Complementarity, CDRs). The network regions adopt a β-leaf conformation and the CDRs can form loops connecting the β-leaf structure. The CDRs in each chain are maintained in their three-dimensional structure by the network regions and form, together with the CDRs of other chains, the antigen binding site. The light and heavy chain CDR3 regions of the antibody play a particularly important role in the specificity / binding affinity of the antibodies according to the invention and therefore provide another objective of the invention.

The terms hypervariate region or antigen-binding portion of an antibody, when used here, refer to amino acid residues of an antibody which are responsible for binding to the antigen. The hypervariable region comprises amino acid residues from the complementarity determining regions or CDRs. Network or RR regions are those regions of variable domain other than the residues of the hypervariable region, as defined here. Therefore, the light and heavy chains of an antibody comprise the N- to the C-terminus of the FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains. In particular, heavy chain CDR3 is the region that contributes the most to antigen binding. CDR and RR regions are determined according to the standard definition of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ^to Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or those residues of a hypervariable loop.

The term nucleic acid or nucleic acid molecule, as used here, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA.

Nucleic acid is operably linked when it is placed in functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosomal binding site is operably linked to a coding sequence if it is positioned to facilitate translation. Generally, operably linked means that the DNA sequences being linked are continuous and, in the case of a secretory leader, continuous and networked. However, intensifiers do not have to be continuous. If such sites do not exist, synthetic oligonucleotide adapters or ligands are used according to conventional practice.

As used herein, the expressions cell, cell line and cell culture are used interchangeably and all such designations include offspring. Thus, the transforming words and transformed cells include the cell in question and cultures derived from it without taking into account the number of transfers. It should also be understood that the entire offspring may not be exactly identical in terms of DNA content, due to deliberate or inadvertent mutations. Variant offspring that have the same biological function or activity, as selected for the originally transformed cell, are included. Where distinct designations are desired, this will be clear from the context.

The constant domains are not directly involved in binding an antibody to an antigen, but exhibit various effector functions. Depending on the sequence of amino acids in the constant region of their heavy chains, antibodies or immunoglobulins are divided into classes: IgA, IgD, IgE, IgG and IgM and several of these can be further divided into subclasses (isotypes), for example, IgG1, IgG2, lgG3 and lgG4, lgA1 and lgA2. The heavy chain constant region that corresponds to the different classes of immunoglobulins is called c, Ô, ε, γ and μ, respectively. The antibodies according to the invention are preferably of the IgG type.

The Fc part of an antibody is directly involved in complement activation, binding to C1q and binding to the Fc receptor. Although the influence of an antibody on the complement system is dependent on certain conditions, binding to C1q is caused by binding sites defined in the Fc part. Such binding sites are known in the art and described, for example, by Boakie et al., Nature 282 (1975) 742-743, Lukas et al., J. Immunol. 127 (1981) 2555-2560, Brunhouse and Cebra, Mol. Immunol. 16 (1979) 907-917, Burton et al., Nature 288 (1980) 338-344, Thommesen et al., Mol. Immunol. 37 (2000) 995-1004, Idusogie et al., J. Immunol.164 (2000) 4178-4184, Hezareh et al., J. Virology 75 (2001) 12161-12168, Morgan et al., Immunology 86 (1995) 319- 324, EP 0307434. Such binding sites are, for example, L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to the North American Kabat index, see below). Antibodies of the subclass lgG1, lgG2 and lgG3 usually show complement activation and binding to C1q and C3, whereas lgG4 does not activate the complement system and does not bind to C1q and C3. As used herein, the term Fc part derived from human origin denotes an Fc part which is an Fc part of a human antibody of the subclass lgG4 or an Fc part of a human antibody of the subclass lgG1, lgG2 or lgG3 which is modified from a such that no link to C1q and / or link to FcR, as defined below, can be detected. The Fc part of an antibody is a term well known to those qualified and defined based on the dividing of antibodies by papain. The antibodies according to the invention contain an Fc part derived from human origin and, preferably, all other parts of the human constant regions. Preferably, the Fc part is a human Fc part and, especially preferably, the human IgG4 subclass or a mutated Fc part of the IgG 1 subclass. Most especially preferred are the Fc parts and the heavy chain constant regions shown in SEQ ID NO: 25-28 or SEQ ID NO: 25 without PVA236 mutation.

II. Preferred embodiments of the invention

The invention comprises an antibody that binds to P-selectin characterized in that the variable heavy chain amino acid sequence CDR3 of said antibody is selected from the group consisting of the heavy chain sequences CDR3 of SEQ ID NO: 38, 39, 40, 41 or 42.

The invention preferably provides an antibody that binds to P-selectin comprising a variable heavy chain and a variable light chain, characterized in that the variable heavy chain comprises CDR sequences CDR1, CDR2 and CDR3 and CDR1 being selected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, CDR2 being selected from the group consisting of SEQ ID NOs: 33, 34, 35, 36, 37, CDR3 being selected from the group consisting of SEQ ID NOs: 38 , 39, 40, 41,42, wherein said CDRs are selected independently of each other.

The antibody according to the invention is preferably characterized by the fact that the variable light chain comprises the CDR sequences CDR1, CDR2 and CDR3 and CDR1 is selected from SEQ ID NOs: 43, 44, CDR2 is selected from SEQ ID NOs: 45, 46 and CDR3 is selected from SEQ ID NOs: 47, 48, 49, 50, 51, 52 in which said CDRs are selected independently of each other.

The antibody is preferably characterized in that it contains, as heavy chain CDRs, the CDRs of SEQ ID NO: 2 and as light chain CDRs, the CDRs of SEQ ID NO: 1, as heavy chain CDRs, the CDRs of SEQ ID NO: 4 and as light chain CDRs, CDRs of SEQ ID NO: 3, as CDRs of heavy chain, CDRs of SEQ ID NO: 6 and as CDRs of light chain, CDRs of SEQ ID NO : 5, as heavy chain CDRs, CDRs of SEQ ID NO: 8 and as light chain CDRs, CDRs of SEQ ID NO: 7, as heavy chain CDRs, CDRs of SEQ ID NO: 10 and as CDRs light chain, the CDRs of SEQ ID NO: 9, as heavy chain CDRs, the CDRs of SEQ ID NO: 12 and as light chain CDRs, the CDRs of SEQ ID NO: 11, as heavy chain CDRs, the CDRs of SEQ ID NO: 14 and as light chain CDRs, CDRs of SEQ ID NO: 13, as CDRs of heavy chain, CDRs of SEQ ID NO: 16 and as CDRs of light chain, CDRs of SEQ ID NO : 15 as heavy chain CDRs, CDRs of SEQ ID NO: 18 and as light chain CDRs, C SEQ ID NO: 17 DRs, as heavy chain CDRs, SEQ ID NO: 20 CDRs and as light chain CDRs, SEQ ID NO: 19 CDRs or as heavy chain CDRs, SEQ ID NO CDRs : 22 and as light chain CDRs, the CDRs of SEQ ID NO: 21.

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..... _.. /. / .: _β · ;:

The CDR sequences can be determined according to the standard definition of Kabat et al, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). CDRs on each chain are separated by a network of amino acids. The CDRs of SEQ ID NO: 1-22 are shown in SEQ ID NO: 29-52.

The antibody according to the invention is preferably characterized by the fact that said antibody binds to P-selectin and comprises a heavy and light variable region independently selected from the group consisting of:

a) the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 2 and the light chain variable domain defined by SEQ ID NO: 1;

b) the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 4 and the light chain variable domain defined by SEQ ID NO: 3;

c) the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 6 and the light chain variable domain defined by SEQ ID NO: 5;

d) the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 8 and the light chain variable domain defined by SEQ ID NO: 7;

e) the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 10 and the light chain variable domain defined by SEQ ID NO: 9;

f) the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 12 and the light chain variable domain defined by SEQ ID NO: 11;

g) the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 14 and the light chain variable domain defined by SEQ IDNO: 13;

h) the heavy chain variable domain defined by the sequence

Μ. ... /. . ·.

amino acids SEQ ID NO: 16 and the light chain variable domain defined by SEQ ID NO: 15;

i) the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 18 and the light chain variable domain defined by SEQ ID NO: 17;

j) the heavy chain variable domain defined by the amino acid sequence SEQ ID NQ: 20 and the light chain variable domain defined by SEQ ID NO: 19;

k) the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 22 and the light chain variable domain defined by SEQ ID NO: 21.

The antibody according to the invention is preferably characterized in that the heavy chain variable region comprises an amino acid sequence independently selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 , 16, 18, 20 and 22.

The antibody according to the invention is preferably characterized in that the light chain variable region comprises an amino acid sequence independently selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 , 15, 17, 19 and 21.

The present invention relates to an antibody that binds to Pselectin and does not bind to complement factor C1q and / or Fc receptor. These antibodies do not stimulate complement-dependent cytotoxicity (GDC) and / or antibody-dependent cell cytotoxicity (ADCC). Preferably, this antibody is characterized by the fact that it binds to P-selectin, contains a human-derived Fc part and does not bind to complement factor C1q. Most preferably, that antibody is a human or humanized antibody.

The antibody according to the invention is preferably characterized by the fact that the constant chains are of human origin. Such constant chains are well known in the art and described, for example, by Kabat (see, for example, Johnson, G. and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218). For example, a useful human heavy chain constant region comprises an amino acid sequence independently selected from the group consisting of SEQ ID NO: 24, 25, 26, 27 and 28. For example, a useful human light chain constant region comprises a amino acid sequence of a kappa light chain constant region of SEQ ID NO: 23.

The effector functions mediated by the Fc part of the Fc region of the antibody refer to effector functions that operate after binding of an antibody to an antigen (these functions involve activation of the complement cascade and / or activation of cells by an Fc receptor (FcR )).

The function of the complement cascade can be assessed by the CH50 assay. Red sheep cells sensitized with anti-red cell (EA) antibodies are added to the test serum to activate the classic route that results in hemolysis. The volume of serum required for lysis of 50% of the red cells determines the CH50 unit. The AP-CH50 measures alternative routes and terminals. The procedure is similar, except that red rabbit cells are used. The alternative route is activated when adding the test serum.

C1q and two serine proteases, C1r and C1s, form the C1 complex, the first component of the complement-dependent cytotoxicity (CDC) pathway. To activate the complement cascade, C1q binds to at least two IgG1 molecules or one IgM molecule, attached to the antigenic target (Ward and Ghetie, Therapeutic Immunology 2: 77-94 (1995)). Burton described (Molec. Immunol., 22 (3): 161-206 (1985)) that the heavy chain region comprising amino acid residues 318 to 337 is involved in complement fixation. Duncan and Winter (Nature 332: 738-40 (1988)), using site-directed mutagenesis, reported that Glu318, Lys320 and Lys322 form the C1q binding site. The role of the Glu318, Lys320 and Lys 322 residues in C1q binding was confirmed by the ability of a short synthetic peptide containing these residues to inhibit complement-mediated lysis.

The term complement-dependent cytotoxicity (CDC) refers to the lysis of human endothelial cells expressing P-selectin and plaqueoZCs by the antibody according to the invention in the presence of complement. CDC is preferably measured by treating human endothelial cells expressing P-selectin and platelets with an antibody according to the invention in the presence of complement. The cells are preferably labeled with calcein. CDC is found if the antibody lyses 20% or more of the target cells at a concentration of 30 pg / ml. However, the inventors have found that, for the properties of the antibodies according to the invention, reduced binding to complement factor C1q in an ELISA assay is essential. In such an assay, in principle, an ELISA slide is coated with antibody concentration ranges, to which purified human C1q or human serum is added. Binding to C1q is detected by an antibody directed against C1q, followed by a peroxidase-labeled conjugate. The binding detection (maximum binding, Bmax) is measured as the optical density at 405 nm (OD405) for the ABTS peroxidase substrate (2,2'-Azino-di- [3-ethylbenzthiazoline-sulfonate (6)]. , the present invention relates to an antibody characterized by the fact that the antibody does not bind to complement factor C1q, which refers to such measurement by an ELISA assay in which the maximum binding (Bmax) of C1q to the antibody at a concentration of 10 pg / ml of the antibody is <30% of the Bmax of the LC 1004-002 antibody of the hu-Mab cell line <Pselectin> LC 1004-002 (DSM ACC2641), preferably 20% or less.

It is further preferred that an antibody according to the invention shows reduced binding to complement factor C3 in an ELISA assay. The test is performed in the same way as the C1q test. In such an assay, in principle, an ELISA slide is coated with antibody concentration ranges, to which purified human C3 or human serum is added. Binding to C3 is detected by an antibody directed against C3, followed by a peroxidase-labeled conjugate. The binding detection (maximum binding, Bmax) is measured as the optical density at 405 nm (OD405) for the ABTS peroxidase substrate (2,2'-Azino-di- [3-ethylbenzthiazoline-sulfonate (6)]. The present invention relates to an antibody characterized by the fact that the antibody does not bind to complement factor C3 which refers to such measurement by an ELISA assay in which the maximum binding (Bmax) of C3 to the antibody at a concentration of 10 pg / ml of the antibody is 10% of the Bmax of the LC 1004-002 antibody of the hu-Mab <P-selectin> cell line LC 1004-002 (DSM ACC2641), preferably 5% or less.

The term antibody-dependent cell cytotoxicity (ADCC) is a function mediated by binding to the Fc receptor and refers to the lysis of target cells that express P-selectin by an antibody according to the invention in the presence of effector cells. ADCC is preferably measured by treating an endothelial cell preparation expressing Pselectin with an antibody according to the invention in the presence of effector cells, such as freshly isolated PBMC (peripheral blood mononuclear cells) or effector cells purified from buffy coat or NK cells (natural killers). Target cells are labeled with ⁵¹ Cr and subsequently incubated with the antibodies. The labeled cells are incubated with effector cells and the supernatant is analyzed for ⁵¹ Cr release. Controls include incubation of the target endothelial cells with effector cells, but without the antibody. The ability of antibodies to induce the initial steps that measure ADCC was determined by measuring their binding to cells expressing Fcy receptors, such as granulocytes (expressing FcyRII and RUI), NK cells (expressing FcyRIII) and monocytes (expressing FcyRI and RH).

Binding of the Fc receptor to effector functions can be mediated by the interaction of the Fc region of an antibody with Fc receptors (FcRs), which are specialized cell surface receptors on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate the removal of antibody-coated pathogens through phagocytosis of immune complexes and the lysis of erythrocytes and various other cellular targets (eg tumor cells) coated with the corresponding antibody, via cytotoxicity anti-dependent cell-mediated (ADCC). Van de Winkel and Anderson, J. Leuk. Biol. 49: 511-24 (1991). FcRs are defined by their specificity by isotypes of immunoglo18 ..... _.. bulin, Fc receptors for IgG antibodies are referred to as FcyR, for IgE as FceR, for IgA as FcocR and so on. Binding to the Fc receptor is described, for example, in Ravetch and Kinet, Ann. Rev. Immunol. 9 (1991) 457-492, Capel et al., Immunomethods 4 (1994) 32-34, de Haas et al., J. Lab. Clin. Med. 126 (1995) 330-341 and Gessner et al., Ann. Hematol. 76 1998) 231-248. The antibodies according to the invention preferably show reduced binding to Fcy receptors, preferably to FycR1, -HA, -IIB and / or IIIA.

The antibodies according to the present invention preferably do not stimulate any effector function and do not bind to the FcyR presented on NK cells. The term non-binding of FcyR, therefore, means that at an antibody concentration of 10 μg / ml, the binding of an antibody according to the invention to NK cells is 1% or less the binding found for the LC 1004 antibody -002 of the hu-Mab <Pselectin> cell line LC 1004-002 (DSM ACC2641).

Although lgG4 shows binding to reduced FcR, antibodies from other IgG subclasses show strong binding. However, Pro238, Asp265, Asp270, Asn297 (Fc carbohydrate loss), Pro329 and 234, 235, 236 and 237 Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 and His435 are residues which also provide, if altered, binding reduced to FcR (Shields et al. J. Biol. Chem. 276 (2001), 6591-6604, Lund et al. FASEB J. 9 (1995), 115-119, Morgan et al., Immunology 86 (1995) 319-324, EP 0307434). Preferably, an antibody according to the invention is, with respect to binding to the FcR, subclass lgG4 or subclass lgG1 or lgG2 with a mutation in S228, L234, L235 and / or D265 and / or contains the mutation PVA236 or GLPSS331 . Especially preferred are the S228P (lgG4), L234A (lgG1), L235A (lgG1), L235E (lgG4), GLPSS331 (lgG1) and / or PVA236 (lgG1) mutations. Preferred combinations of mutations are also shown in Table 1. An additional preferred combination is D265A / N297A.

The term that binds to P-selectin, as used here, means that binding of the antibody to P-selectin in a BIAcore assay (Pharmacia Biosensor AB, Uppsala, Sweden) or in an ELISA in which purified P-selectin cZ ^ or CHO / P-selectin transfectants are coated on microtiter slides.

In the BIAcore assay, the antibody is bound to a surface and the binding of P-selectin is measured using Plasmon Resonance on 5 Surface (SPR). The binding affinity is defined by the terms ka (rate constant for the association of the antibody from the antibody / antigen complex), kd (dissociation constant) and K _D (kd / ka). The antibodies according to the invention show a K _D of 10 ' ⁸ or less, preferably about 10 ^{11 to} 10 ⁹ M (see examples). Consequently, the present invention relates to an antibody as described above in which the antibody binds to P-selectin with a KD value of less than 10 ' ⁸ M in a BIAcore assay, preferably where the KD range is from 10 ^_11 to 10 ⁹ M.

Preferably, the antibody is of the human IgG1 or IgG4 subtype.

More preferably, the antibody is characterized in that the antibody is an antibody of the human lgG1 subclass, containing at least one mutation in L234, L235, D270, N297, E318, K320, K322, P331 and / or P329 or an antibody of the subclass human IgG4, containing at least one mutation in L235 and S228 (numbered according to the North American index).

^{20 In the} P-selectin-specific ^ELISA , purified P-selectin is coated on microtiter slides and antibody binding to P-selectin is detected with a biotinylated anti-human IgG and the usual ELISA steps. The EC50 values in that assay preferably range between 0.002 and 0.03 pg / ml on CHO-P-selectin cells, that is, the present invention relates to antibodies in which the EC50 values for Pselectin binding are in the range of 0.002 to 0.03 pg / ml on CHO cells showing P-selectin in an ELISA assay. In an assay in which CHO transfectants expressing P-selectin are coated on the microtiter slide, EC50 values range between 0.01 and 0.08 pg / ml, preferably 30 between 0.01 and 0.04 pg / ml .

EC _S0 values for E- and L-selectin transfectants are preferably above 100 pg / ml. The antibodies of the present invention

3> o ²⁰ ··,:. ·· ♦ *: **.

are characterized by the fact that they bind at least 1000 times more specifically to P-selectin than to E- and / or L-selectin as measured by the EC50 values in an ELISA assay, where P- and E- and / or Lselectin are coated on the microtiter slide.

The term inhibition of binding of the P-selectin ligand to Pselectin, as used herein, refers to the binding of purified or cell-expressed P-selectin to its ligand shown on HL60 cells. The binding of P-selectin to its ligand is inhibited by the antibodies according to the invention. Inhibition is measured as IC 50 in in vitro assays that analyze the antibody's ability to inhibit the binding of P-selectin to a ligand. Such assays are described in the Examples. They use, as suitable sources of P-selectin, affinity-purified P-selectin and activated platelets and, as suitable sources of the ligand, leukocyte-like cells, such as HL60 cells, in such adhesion assays to HL60 cells, expressing PSGL- 1 as the physiologically relevant linker of P-selectin, to P-selectin or activated platelets is measured without and with increasing concentrations of the antibody. IC50 values are measured as mean values from at least three independent measurements. Inhibition means an IC ₅₀ value of no more than 1 pg / ml, preferably 0.5 to 0.08 pg / ml.

The antibodies of the present invention inhibit the adhesion of leukocyte-like HL60 cells to purified P-selectin with IC 50 values in the range of 0.08 to 0.5 pg / ml, preferably 0.08 to 0.11 pg / ml. Adhesion of leukocyte-like HL60 cells to activated platelets is inhibited with IC 50 values in the range of 0.05 to 0.3 pg / ml.

Consequently, other modalities of the present invention refer to antibodies characterized by the fact that the EC50 values for binding to P-selectin are in the range of 0.01 to 0.08 pg / ml in an ELISA assay in which CHO transfectants expressing P- selectin are coated on the microtiter slide. The preferred range is 0.01 to 0.04 pg / ml. EC50 values on E- and L-selectin transfectants are above 100 pg / ml. In another embodiment, the antibodies of the present invention inhibit the adhesion of leukocyte-like HL60 cells to P-selectin

3 /

purified with IC50 values between 0.08 to 0.5 pg / ml The preferred range is 0.08 to 0.11 gg / ml.

The antibodies of the present invention inhibit the interaction of leukocytes with a platelet monolayer, preferably by more than 70% in a fully human flow system (at a concentration of 10 pg / ml). In addition, these antibodies inhibit leukocyte adhesion to activated endothelial cells in a human flow system in the range of 60-90% at a concentration of 3 μ ₉ / πΊΐ (with differential effects on leukocyte subtypes).

The antibodies of the present invention are preferably capable of binding to P-selectin in the presence of aa fragment of P-selectin from aa 60-75 (sequence in Swiss-Prot P16109) and / or do not competitively inhibit the binding of a antibody secreted by a cell line called ATCC Accession No. HB11041 to P-selectin.

The antibodies of the invention preferably do not inhibit the interaction of P-selectin with the platelet membrane glycoprotein GPIba in an ELISA assay format. In the ELISA glycocalycin, the soluble extracellular part of the GPIba was immobilized on the microtiter slide wells, as described (Romo et al., J Exp Med 190: 803 (1999) and the purified P-selectin binding after pre-incubation with the HuMabs to Pselectin was detected with a polyclonal anti-P-selectin antibody.

In another preferred embodiment of the present invention, the antibody characterized by the fact that it does not bind to the C3 protein is, more preferably, characterized by the fact that it does not stimulate complement-dependent cytotoxicity (CDC). In addition, the antibody can be characterized by the fact that it does not bind to Fcy receptors on NK effector cells. Preferably, the antibody is characterized in that it is an antibody of the human IgG1 subclass, containing at least one mutation in L234, L235, D270, N297, E318, K320, K322, P331 and / or P329 or an antibody of the lgG4 subclass human, containing at least one mutation in L235 and S228 (numbered according to the EU index). In another preferred embodiment, the antibody is characterized by the fact that it does not stimulate antibody-dependent cell cytotoxicity (ADCC).

In an even more preferred embodiment, the antibodies of the present invention are characterized by the fact that they bind to P-selectin and comprise a variable region independently selected from the group consisting of:

a) the light chain variable domain defined by the amino acid sequence SEQ ID NO: 1 and the heavy chain variable domain defined by SEQ IDNO: 2;

b) the light chain variable domain defined by the amino acid sequence SEQ ID NO: 3 and the heavy chain variable domain defined by SEQ ID NO: 4;

c) the light chain variable domain defined by the amino acid sequence SEQ ID NO: 5 and the heavy chain variable domain defined by SEQ ID NO: 6;

d) the light chain variable domain defined by the amino acid sequence SEQ ID NO: 7 and the heavy chain variable domain defined by SEQ ID NO: 8;

e) the light chain variable domain defined by the amino acid sequence SEQ ID NO: 9 and the heavy chain variable domain defined by SEQ ID NO: 10;

f) the light chain variable domain defined by the amino acid sequence SEQ ID NO: 11 and the heavy chain variable domain defined by SEQ ID NO: 12;

g) the light chain variable domain defined by the amino acid sequence SEQ ID NO: 13 and the heavy chain variable domain defined by SEQ ID NO: 14;

h) the light chain variable domain defined by the amino acid sequence SEQ ID NO: 15 and the heavy chain variable domain defined by SEQ ID NO: 16;

i) the light chain variable domain defined by the amino acid sequence SEQ ID NO: 17 and the heavy chain variable domain defined by SEQ ID NO: 18;

.........

j) the light chain variable domain defined by the amino acid sequence SEQ ID NO: 19 and the heavy chain variable domain defined by SEQ ID NO: 20; and

k) the light chain variable domain defined by the amino acid sequence SEQ ID NO: 21 and the heavy chain variable domain defined by SEQ IDNO: 22.

Preferably, the antibodies comprise the light chain variable domain defined by the amino acid sequence SEQ ID NO: 3 and the heavy chain variable domain defined by SEQ ID NO: 4.

Preferred antibodies are characterized by the fact that the antibodies are of the human IgG4 subclass or comprise at least one amino acid mutation that causes non-binding to complement factor C1q. Such variant antibodies comprise, for example, the amino acid sequence independently selected from the group consisting of SEQ ID NO: 25 or SEQ ID NO: 26 and SEQ ID NO: 28.

A variant anti-P-selectin antibody refers here to a molecule which differs, in terms of the amino acid sequence, from an amino acid sequence of the original anti-P-selectin antibody by virtue of the addition, deletion and / or substitution of one or more more amino acid residues in the original antibody sequence. In the preferred embodiment, the variant comprises one or more amino acid substitutions in one or more constant or variable regions of the original antibody, preferably in the constant region. For example, the variant may comprise at least one, for example, from about one to about ten and, preferably, from about two to about five substitutions in one or more variable regions of the original antibody. Commonly, the variant will have an amino acid sequence having at least 90% amino acid sequence identity with the constant and / or variable domain sequences of the original antibody, more preferably at least 95% and even more preferably at least 99%.

Identity or homology with respect to this sequence is defined here as the percentage of amino acid residues in the candi24 data sequence that are identical to the residues in the original antibody, after aligning the sequences and introducing intervals, if necessary, to obtain maximum sequence percent identity . None of C-terminal, N-terminal or internal extensions, deletions or insertions in the antibody sequence should be constructed as affecting the identity or sequence homology. The variant retains the ability to bind to human P-selectin and, preferably, has properties which are superior to those of the original antibody. For example, the variant may have a stronger binding affinity, an enhanced ability to treat a disease associated with critical limb ischemia or occlusive disease of peripheral arteries (CLI / PAOD).

The variant antibody of particular interest here is one which shows at least about 4-fold enhancement in inhibitory activity in an adhesion assay when compared to the original antibody due to the elimination of binding to Fcy receptors.

The original antibody here is one which is encoded by an amino acid sequence used to prepare the variant. Preferably, the original antibody has a human-based region and, if present, has human antibody constant regions. For example, the original antibody can be a humanized or human antibody.

Antibodies according to the invention further include such antibodies having conservative sequence modifications, nucleotide and amino acid sequence modifications which do not alter or affect the above mentioned characteristics of the antibody according to the invention. Modifications can be introduced using standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which the amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine , threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and chains aromatic sides (eg tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue provided for in an anti-P-selectin antibody can preferably be replaced by another amino acid residue from the same side chain family.

Amino acid substitutions can be performed through mutagenesis based on molecular modeling, as described by Riechmann, L. and others, Nature 332 (1988) 323-327 and Queen, C. and others, Proc. Natl. Acad. Know. USA 86 (1989) 10029-10033.

In another preferred embodiment, the antibodies comprise the k light chain constant region, as defined by SEQ ID NO: 23.

Preferred antibodies according to the invention are antibodies defined as lgG1v1 (PVA-236; GLPSS331 as specified by E233P; L234V; L235A; delta G236; A327G; A330S; P331S), lgG1v2 (L234A; L235A) and lgG2v1 (L228); .

In another preferred embodiment, these antibodies also comprise antibody fragments selected from the group consisting of Fab, F (ab ') ₂ and single-stranded fragments.

The invention further comprises a method for producing an antibody according to the invention comprising the steps of a) transforming a host cell with a first nucleic acid sequence encoding the light chain of an original human antibody according to the invention and a second DNA sequence encoding the heavy chain of said original human antibody, wherein the Fc part is modified by the fact that said Fc part does not bind to complement factor C1q and / or Fc receptor; b) expressing said first and second DNA sequences so that said antibody and light chains are produced; and c) recovering said host cell antibody or host cell culture.

The invention also relates to intermediate antibodies, that is, anti-P-selectin antibodies characterized by the fact that these antibodies are human or humanized antibodies and bind at least 1000 times more specifically to P-selectin than to E- or L- selectin, as measured in an ELISA assay in which P- and E- and / or L-selectin are coated on the microtiter slide. Preferably, these antibodies are antibodies to IgG1 or IgG4. These antibodies can also comprise the amino acid sequence as defined by the γ1 heavy chain constant region in SEQ ID NO: 24 or the γ4 heavy chain constant region in SEQ ID NO: 27. In particular, these antibodies refer to antibodies produced by a cell line selected from the group consisting of hu-Mab <P-selectin> LC 1004-001 (DSM ACC2640), huMab <P-selectin> LC 1004-002 (DSM ACC2641) and hu-Mab <P-selectin> LC 1004 -017 (DSM ACC2642).

The antibodies according to the invention include, in addition, such antibodies having conservative sequence modifications, nucleotide and amino acid sequence modifications which do not affect or alter the above mentioned characteristics of the antibody according to the invention. Modifications can be introduced using standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which the amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been identified in the art. These families include amino acids with basic side chains (for example, lysine, arginine, histidine), acidic side chains (for example, aspartic acid, glutamic acid), uncharged polar side chains (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, phenylalanine, methionine), beta-branched side chains (for example, threonine, valine, isoleucine) and aromatic side chains (for example, tyrosine, phenylalanine, tripe31

........

pane, histidine). Thus, a non-essential amino acid residue predicted on an anti-human P-selectin antibody can preferably be replaced by another amino acid residue from the same side chain family.

Amino acid substitutions can be performed through mutagenesis based on molecular modeling, as described by Riechmann, L. and others, Nature 332 (1988) 323-327 and Queen, C. and others, Proc. Natl. Acad. Know. USA 86 (1989) 10029-10033.

The present invention also comprises nucleic acid molecules that encode an antibody mentioned above, the corresponding vectors comprising those nucleic acids and the corresponding host cell for those vectors. The invention encompasses a method for preparing the antibodies comprising culturing the corresponding host cells under conditions that permit the synthesis of said antibody molecules and the recovery of said antibodies from said culture, for example, through expression of a nucleic acid encoding the chain heavy and a nucleic acid encoding the light chain in a prokaryotic or eukaryotic host cell and recovering said polypeptide from said cell.

Diagnostic and therapeutic uses for the antibody are considered. In a diagnostic application, the invention provides a method for determining the presence of the P-selectin protein comprising exposing a sample suspected to contain P-selectin to the antiP-selectin antibody and determining the binding of the antibody to the sample. For that use, the invention provides a kit comprising the antibody and instructions for using the antibody to detect the P-selectin protein.

The antibodies of the present invention are useful for the treatment of inflammatory and thrombotic diseases. Such diseases include vascular disorders, such as atherosclerosis, venal and deep arterial thrombosis, restenosis after angioplasty or stent placement. Preferred applications are peripheral artery occlusive disease (PAOD) and critical limb ischemia (CLI). Other applications are the treatment of post-ischemic leukocyte-mediated tissue damage caused by myocardial infarction, cerebral ischemic events (eg, stroke), renal infarction and the like. The antibodies are also suitable for the treatment of sepsis, acute leukocyte-mediated lung injury and allergic reactions, such as asthma. Other applications are the prevention of organ transplant rejection and autoimmune diseases, including rheumatoid arthritis. In addition, tumor metastasis can be prevented by inhibiting the adhesion of circulating cancer cells.

The invention further provides a method for the treatment of a mammal that is suffering from the aforementioned thrombotic and inflammatory disorders, especially PAOD and CLI (peripheral artery occlusive disease or critical limb ischemia).

The invention further provides for the use of the above antibodies for therapy, for example, for the manufacture of medicaments for the treatment of these diseases.

The invention relates to the use of antibodies as defined above for the manufacture of a pharmaceutical composition and comprises a pharmaceutical composition containing an antibody according to the invention with a pharmaceutically effective amount, optionally together with a buffer and / or adjuvant useful for the formulation of antibodies for pharmaceutical purposes.

The invention further provides pharmaceutical compositions comprising such antibodies in a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition can be included in a manufacturing article or kit.

The invention further provides hybridoma cell lines which produce antagonistic monoclonal antibodies, for example, the original antibodies, according to the invention.

The preferred hybridoma cell lines according to the invention, hu-Mab <P-selectin> LC 1004-001 (HuMab 001 antibody) huMab <P-selectin> LC 1004-002 (HuMab 002 antibody) and hu-Mab < Pselectin> LC 1004-017 (HuMab 017 antibody) were deposited, under the Budapest Treaty, with international recognition of deposit of <3θ

........... · ··:

microorganisms for the purpose of patent procedure, at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Germany:

Cell line Deposit No. Deposit Date hu-Mab <P-selectin> LC 1004-001 DSM ACC2640 03/30/2004 hu-Mab <P-selectin> LC 1004-002 DSM ACC2641 03/30/2004 hu-Mab <P-selectin> LC 1004-017 DSM ACC2642 03/30/2004

Antibodies obtainable from said cell lines are preferred embodiments of the invention.

The antibodies according to the invention are preferably produced using recombinant means. Such methods are widely known in the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody polypeptide and, usually, purification to a pharmaceutically acceptable purity. For protein expression, nucleic acids encoding light and heavy chains or fragments thereof are inserted into expression vectors using standard methods. Expression is performed on appropriate prokaryotic or eukaryotic host cells, such as CHO cells, NSO cells, SP2 / 0 cells, HEK293 cells, COS cells, yeast cells or E. coli and the antibody is recovered from the cells (supernatant or cells after lysis).

Recombinant antibody production is well known in the art and described, for example, in the reviewed articles by Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S. et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R.J., Mol. Biotechnol. 16 (2000) 151-161; Werner, R.G., Drug Res. 48 (1998) 870-880.

The antibodies can be present in whole cells, in a cell lysate or in a substantially pure or partially purified form. Purification is performed in order to eliminate other cellular components or other contaminants, for example, other nucleic acids or proteins, using standard techniques, including alkaline / SDS treatment, column chromatography and others well known in the art. See Ausubel, F. et al., Ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

Expression in NSO cells is described, for example, by Barnes, L.M. and others, Cytotechnology 32 (2000) 109-123; and Barnes, LM et al., Biotech. Bioeng. 73 (2001) 261-270, transient expression is described, for example, by Durocher, Y. and others, Nucl. Acids. Res. 30 (2002) E9. Cloning of variable domains is described by Orlandi, R. et al., Proc. Natl. Acad. Know. USA 86 (1989) 3833-3837; Carter, P. et al., Proc. Natl. Acad. Know. USA 89 (1992) 4285-4289; and Norderhaug, L. et al., J. Immunol. Methods 204 (1997) 77-87. A preferred transient expression system (HEK 293) is described by Schlaeger, E.-J. and Christensen, K „in Cytotechnology 30 (1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996) 191-199.

Control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence and a ribosomal binding site. Eukaryotic cells are known to use promoters, enhancers and polyadenylation signals.

Nucleic acid is operably linked ”when it is placed in a functional relationship with another sequence of nucleic acid. For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosomal binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, operably linked means that the DNA sequences being linked are continuous and, in the case of a secretory leader, continuous and networked. However, intensifiers do not have to be continuous. The connection is carried out by connecting at suitable restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or ligands are used according to conventional practice.

Monoclonal antibodies are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. DNA and RNA encoding monoclonal antibodies are readily isolated and sequenced using conventional procedures. Hybridoma cells can serve as a source of such DNA and RNA. Once isolated, DNA can be inserted into expression vectors which are then transfected into host cells, such as HEK 293 cells, CHO cells or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of recombinant monoclonal antibodies in host cells.

Amino acid sequence variants (or mutants) of an anti-human P-selectin antibody are prepared by introducing appropriate nucleotide changes into the antibody's DNA or by nucleotide synthesis. Such modifications can be made, however, only in a very limited range, for example, as described above. For example, the modifications do not alter the characteristics of the aforementioned antibody, such as the IgG isotype and binding, but can improve the yield of recombinant production, protein stability or facilitate purification.

Any cysteine residue not involved in maintaining the proper conformation of the anti-P-selectin antibody can also be replaced, usually with serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine binding (s) can be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment, such as an Fv fragment).

Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody. By alteration is meant deletion of one or more carbohydrate moieties found in the antibody and / or addition of one or more glycosylation sites that are not present in the antibody. Antibody glycosylation is typically N-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The asparagine-X-serine and asparagine-X-threonine tripeptide sequences, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate portion to the asparagine side chain. Thus, the presence of these tripeptide sequences in a polypeptide creates a potential glycosylation site. The addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence, so that it contains one or more of the tripeptide sequences described above (for linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of anti-P-selectin antibodies are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation through oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis and mutagenesis in cassette of a previously prepared variant or a non-variant version of humanized anti-Pselectin antibody.

The invention also relates to immunoconjugates comprising the antibody according to the invention conjugated to a cytotoxic agent, such as a chemotherapeutic agent, toxin (for example, an emphatically active toxin of bacterial, fungal, plant or animal origin or fragments thereof) ), a radioactive isotope (i.e., a radioconjugate) or a prodrug of an agent for the prophylaxis or treatment of thrombotic and inflammatory disorders, especially of PAOD and CLI. Antibody and cytotoxic agent conjugates are made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional imidoester derivatives; (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis (p-azidobenzoyl) hexanediamine), bisΫ3 diazonium derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-di nitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta, E.S. and others, Science 238 (1987) 1098-1104). Labeled 1-isothiocyanatobenzyl-3-methylldiethylene triaminapentaacetic acid (MX-DTPA) carbon-14 is an exemplary chelating agent for the conjugation of radionuclide to the antibody. See WO 94/11026.

Another type of covalent modification involves chemical or enzymatic coupling of glycosides to the antibody. These procedures are advantageous in that they do not require the production of the antibody in a host cell that has glycosylation capabilities for Nou O-linked glycosylation. Depending on the coupling mode used, the sugar (s) can (s) be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as those from cysteine, (d) free hydroxyl groups, such as those from serine, threonine or hydroxyproline, (e) aromatic residues, such as those from phenylalanine, tyrosine or tryptophan, or (f) the amide group from glutamine. These methods are described in WO 87/05330 and in Aplin, J.D. and Wriston, J.C. Jr., CRC Crit. Rev. Biochem. (1981) 259-306.

Removal of any carbohydrate portions present in the antibody can be performed chemically or enzymatically. Chemical deglycosylation requires exposure of the antibody to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the dividing of most or all sugars, except binding sugar (Nacetylglycosamine or N-acetylgalactosamine), while leaving the antibody intact. Chemical deglycosylation is described by Sojahr, H.T. and Bahl, O.P., Arch. Biochem. Biophys. 259 (1987) 52-57 and by Edge, A.S. and others Anal. Biochem. 118 (1981) 131-137. Enzymatic dividing of carbohydrate moieties over antibodies can be achieved through the use of a variety of endo- and exo-glycosidases, as described by Thotakura, N.R. and Bahl, O.P., Meth. Enzymol. 138 (1987) 350-359.

Another type of covalent antibody modification comprises

7?

³⁴ : ·. : · * ♦ · '· · * · · / • * * »· 4 · · * * * * binding of the antibody to one of a variety of non-proteinaceous polymers, for example, polyethylene glycol, polypropylene glycol or polyoxyalkylene, from manner disclosed in US Patent Nos. 4,640,835; 4,496,689; 4,301. 144; 4,670,417; 4,791,192 or 4,179,337.

In yet another aspect, the invention provides B cells isolated from a transgenic non-human animal, for example, a transgenic mouse, which expresses anti-human P-selectin antibodies (for example, the original antibodies produced by a selected cell line of the group consisting of hu-Mab <P-selectin> LC 1004-001 (DSM ACC2640), hu-Mab <P-selectin> LC 1004-002 (DSM ACC2641) and huMab <P-selectin> LC 1004-017 (DSM ACC2642) according to the invention KM mice are suitable trans-chromosomal mice. The KM mouse contains a human heavy chain transchromosome and a human kappa light chain transgene. Endogenous mouse light and heavy chain genes have also been disrupted in KM mice. , so that immunization of mice leads to the production of human immunoglobulins instead of mouse immunoglobulins Construction of KM mice and their use to stimulate human immunoglobulins are described in detail in WO 02 / 43478.

Preferably, the isolated B cells are obtained from a transgenic non-human animal, for example, a transgenic mouse, which has been immunized with a purified or recombinant form of antigen to P-selectin and / or cells that express P- selectin. Preferably, the transgenic non-human animal, for example, a transgenic mouse, has a genome comprising a human heavy chain transgene and a human light chain transgene that encodes all or part of an antibody of the invention. The isolated B cells are then immortalized to provide a source (for example, hybridoma) of anti-human Pselectin antibodies. Accordingly, the present invention also provides a hybridoma capable of producing human monoclonal antibodies according to the invention. In one embodiment, the hybridoma includes a B cell obtained from a transgenic non-human animal, for example, a transgenic dong mouse, having a genome comprising a human heavy chain transgene and a human light chain transgene encoding all or part of an antibody of the invention, fused to an immortalized cell.

In a particular embodiment, the transgenic non-human animal is a transgenic mouse having a genome comprising a human heavy chain transgene and a human light chain transgene that encodes all or part of an antibody of the invention. The transgenic non-human animal can be immunized with a purified preparation 10 or enriched with antigen to P-selectin and / or cells that express Pselectin. Preferably, the transgenic non-human animal, for example, the transgenic mouse, is capable of producing P-selectin isotypes of human monoclonal antibodies to P-selectin.

Human monoclonal antibodies according to the invention can be produced by immunizing a transgenic non-human animal, for example, a transgenic mouse, having a genome comprising a human heavy chain transgene and a human light chain transgene encoding the entire or part of an antibody of the invention, with a preparation enriched or purified from antigen to P-selectin 20 and / or cells expressing P-selectin. Animal B cells (e.g., splenic B cells) are then obtained and fused with myeloma cells to form immortal hybridoma cells that secrete human monoclonal antibodies against P-selectin.

In a preferred embodiment, human monoclonal antibodies directed against P-selectin can be generated using transgenic mice bringing parts of the immune system instead of the mouse system. These transgenic mice, referred to here as HuMab mice, contain a human immunoglobulin gene minilocal that encodes untidy human immunoglobulin genes which include heavy chains (μ and γ) and κ light chain (constant region genes), along with targeted mutations that inactivate the site of the endogenous μ and κ chains (Lonberg, N. et al., Nature 368 (1994) 856-859). Consequence36

the mice exhibit reduced expression of mouse IgM or K in response to immunization, introduced human light and heavy chain transgenes undergo class switching and somatic mutation to generate high-affinity human IgG monoclonal antibodies (Lonberg, N. and others, Nature 368 (1994) 856-859; reviewed in Lonberg, N., Handbook of Experimental Pharmacology 113 (1994) 49-101; Lonberg, N. and Huszar, D., Intern. Rev. Immunol. 25 (1995) 65-93; and Harding, F. and Lonberg, N., Ann. N. Acad. Sci 764 (1995) 536-546). The preparation of HuMab mice is described in Taylor, L. et al., Nucleic Acids Research 20 (1992) 6287-6295; Chen, J. et al., International Immunology 5 (1993) 647-656; Tuaillon, N. et al., Proc. Natl. Acad. Sci USA 90 (1993) 3720-3724; Choi, T.K. and others, Nature Genetics 4 (1993) 117-123; Chen, J. et al., EMBO J. 12 (1993) 821-830; Tuaillon, N. et al., Immunol. 152 (1994) 2912-2920; Lonberg, N. et al., Nature 368 (1994) 856-859; Lonberg, N., Handbook of Experimental Pharmacology 113 (1994) 49-101; Taylor, L. et al., Int. Immunol. 6 (1994) 579-591; Lonberg, N. and Huszar, D., Intern. Rev. Immunol. 25 (1995) 65-93; Harding, F. and Lonberg, N., Ann. N. Acad. Sci 764 (1995) 536546; Fishwild, D.M. and others, Nat. Biotechnol. 14 (1996) 845-851, the contents of all of which are incorporated herein by reference in their entirety. See also US Patents Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877. 397; 5,661,016; 5,814,318; 5,874,299; 5,545,807; 5,770,429; WO 98/24884; WO 94/25585; WO 93/1227;

WO 92/22645; and WO 92/03918.

To generate fully human monoclonal antibodies to Pselectin, HuMab mice can be immunized with a purified or antigen enriched preparation to P-selectin and / or cells expressing P-selectin according to the general method as described by Lonberg, N. and others , Nature 368 (1994) 856-859; Fishwild, D.M. and others, Nat Biotechnol. 14 (1996) 845-851 and WO 98/24884. Preferably, the mice will be 6-16 weeks old when they first immunize. For example, a purified or soluble antigen preparation to P-selectin (for example, purified from cells expressing P-selectin) can be used to immunize HuMab mice intraperitoneally. In the event that immunizations using a purified or antigen enriched preparation to P-selectin do not result in antibodies, mice can also be immunized with cells expressing P-selectin, for example, a tumor cell line, to promote immune responses. Cumulative experience with various antigens has shown that transgenic HuMab mice respond best when initially immunized intraperitoneally (i.p.) with antigen in Freund's complete adjuvant, followed by i.p. every other week (for example, up to a total of 6) with antigen in incomplete Freund's adjuvant. The immune response can be monitored during the course of the immunization protocol with plasma samples being obtained through retroorbital blood collection. Plasma can be selected by ELISA and mice with sufficient titers of human anti-P-selectin immunoglobulin can be used to immortalize the corresponding B cells. Mice can be boosted intravenously with antigen 3 to 4 days before sacrifice and removal of the spleen and lymph nodes. It is expected that 2-3 fusions for each antigen may be required. Several mice will be immunized for each antigen. For example, a total of twelve HuMab mice of the genera HCo7 and HCo12 can be immunized.

HCo7 mice have a JKD break in their endogenous light chain (kappa) genes (as described in Chen, J. et al., EMBO J. 12 (1993) 821-830), a CMD break in their endogenous heavy chain genes (as described in Example 1 of WO 01/14424), a KCo5 human kappa light chain transgene (as described in Fishwild, DM et al., Nat. Biotechnol. 14 (1996) 845-851) and a human heavy chain transgene HCo7 (as described in US Patent No. 5,770,429).

HCo12 mice have a JKD disruption in their endogenous light chain (kappa) genes (as described in Chen, J. et al., EMBO J. 12 (1993) 821-830), a CMD disruption in their endogenous heavy chain genes (as described in Example 1 of WO 01/14424), a KCo5 human kappa light chain transgene (as described in Fishwild, DM et al., Nat. Biotechnol. 14 (1996) 845-851) and a human heavy chain transgene HCo12 (as described in Example 2 of WO 01/14424). Mouse lymphocytes can be isolated and fused with a mouse myeloma cell line using standard PEG-based protocols to generate hybridomas. The resulting hybridomas are then selected with respect to the production of antigen-specific antibodies. For example, single-cell lymphocyte-derived and splenic lymph cell suspensions from immunized mice are fused to one-sixth of the number of mouse myeloma cells that do not secrete SP 2/0 (ATCC, CRL 1581) with 50% PEG . The cells are placed at approximately 2 x 10 ⁵ on a flat-bottomed microtiter slide, followed by incubation for about 2 weeks in selective media.

Individual wells are then selected by ELISA for anti-human P-selectin IgG and IgM monoclonal antibodies. Once extensive hybridoma growth occurs, the medium is analyzed, usually after 10-14 days. Antibody-secreting hybridomas are replaced on slides, selected again and, if still positive for human IgG, anti-P-selectin monoclonal antibodies can be subcloned at least twice by limiting dilution. The stable subclones are then cultured in vitro to produce antibody in tissue culture medium for characterization.

Because CDR sequences are responsible for antibody-antigen interactions, it is possible to express recombinant antibodies according to the invention by constructing expression vectors that include the CDR sequences according to the invention on base sequences from of a different human antibody (see, for example, Riechmann, L. et al., Nature 332 (1998) 323-327; Jones, P. et al., Nature 321 (1986) 522-525; and Queen, C. and others , Proc. Natl. Acad. Sci. USA 86 (1989) 10029-10033). Such base strings can be obtained from public DNA databases that include strings of ³⁹ : * ·:: í * ·.

germline human antibody gene. These germline sequences will differ from the mature antibody gene sequences in that they will not include fully assembled variable genes, which are formed through V (D) J union during B cell maturation. Germline gene sequences will also differ from the sequences of an antibody secondary repertoire of high affinity in individuals uniformly across the variable region.

The invention further comprises the use of an antibody according to the invention for the diagnosis of P-selectin in vitro, preferably through an immunological assay that determines the binding between the Pselectin of a sample and the antibody according to the invention.

In another aspect, the present invention provides a composition, containing one or a combination of human monoclonal antibodies or the antigen binding portion thereof of the present invention, formulated together with a pharmaceutically acceptable carrier. More specifically, the composition is a pharmaceutical or diagnostic composition and even more specifically the pharmaceutical composition comprises an antibody as defined above and at least one pharmaceutically acceptable excipient.

The pharmaceutical compositions of the invention can also be administered in combination therapy, that is, in combination with other agents. For example, the combination therapy may include a composition of the present invention with at least one agent useful in the prophylaxis or treatment of a disease associated with critical limb ischemia (CLI / PAOD) or other conventional therapy.

As used herein, pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, absorption delay and isotonic agents and the like that are physiologically compatible. Preferably, the vehicle is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or transdermal administration (for example, by injection or infusion).

A pharmaceutically acceptable salt refers to a salt that retains the biological activity of the antibody and does not confer any undesired toxicological effects (see, for example, Berge, SM, et al., J. Pharm. Sci. 66 (1977) 1-19 ). Such salts are included in the invention. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids, such as hydrochloric salts.

A composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by those qualified, the route and / or mode of administration will vary, depending on the desired results.

To administer a compound of the invention through certain routes of administration, it may be necessary to coat the compound with or co-administer the compound with a material that prevents its inactivation. For example, the compound can be administered to an individual in an appropriate vehicle, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffered solutions.

Pharmaceutically acceptable excipients or vehicles include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such means and agents for pharmaceutically active substances is known in the art.

The terms parenteral and parenterally administered, as used herein, mean modes of administration other than enteral and topical administration, usually by injection and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intra-orbital injection and infusion. , intracardiac, intradermal, intraperitoneal, transtraqueal, subeutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intra-spinal, epidural and intrasternal.

Such compositions may also contain excipients or adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured through sterilization procedures, above, and through the inclusion of various anti-bacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, ascorbic acid and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be achieved by including agents which delay absorption, such as aluminum monostearate and gelatin.

Regardless of the route of administration selected, the compounds of the present invention, which can be used in a suitable hydrated form and / or the pharmaceutical compositions of the present invention are formulated in pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. technical.

The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied in order to obtain an amount of the active ingredient which is effective in obtaining the desired therapeutic response for a particular patient, composition and mode of administration, without being toxic to the patient. The dosage level selected will depend on a variety of pharmacokinetic factors, including the activity of the compositions of the present invention employed in particular, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the compound being used in particular, duration of treatment, other drugs, compounds and / or materials used in combination with the compositions used in particular, the age, sex, weight, condition, general health and past medical history of the patient being being treated and similar factors well known in the art. A typical weekly dosage could range from about 0.1 mg / kg to about 20 mg / kg or more, depending on the factors mentioned above.

The composition must be sterile and fluid to the extent that the composition is capable of distribution via a syringe. In addition to AS2 ^{42: :::} :: ^·::

water, the vehicle can be an isotonic buffered saline solution, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol and the like) and suitable mixtures thereof.

Appropriate fluidity can be maintained, for example, through the use of a coating, such as lecithin, through maintaining the required particle size in the case of dispersion and through the use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of injectable compositions can be achieved by including, in the composition, an agent which delays absorption, for example, aluminum monostearate or gelatin.

The invention comprises a method for treating a patient in need of therapy characterized by administering to the patient a therapeutically effective amount of an antibody which binds to P-selectin, contains an Fc part derived from human origin and is not binds to the complement factor C1q.

The invention comprises the use of an antibody which binds to P-selectin, contains an Fc part derived from human origin and does not bind to complement factor C1q for therapy.

The invention comprises the use of an antibody which binds to P-selectin, contains an Fc part derived from human origin and does not bind to complement factor C1q for the preparation of a medicine for the prophylaxis and treatment of thrombotic and inflammatory disorders .

The invention comprises the use of an antibody which binds to P-selectin, contains an Fc part derived from human origin and does not bind to complement factor C1q for the treatment of PAOD and CLI.

The present invention thus provides an antibody that binds to P-selectin, does not bind to complement factor C1q, containing an Fc part derived from human origin and being characterized by the fact that said antibody is an antibody of the human lgG1 subclass. , containing at least one mutation in L234, L235, D270, N297, E318, K320, K322, P331 and / or P329 or be an antibody of the human lgG4 subclass in which

S228 is replaced by P and L235 is replaced by E. In one embodiment, the antibody is a human antibody. In another embodiment, the antibody is a humanized antibody.

In one embodiment, the present invention provides an antibody that binds to P-selectin, does not bind to complement factor C1q, containing an Fc part derived from human origin and being characterized by the fact that said antibody is an antibody of the IgG1 subclass human, containing at least one mutation in L234, L235, D270, N297, E318, K320, K322, P331 and / or P329 or being an antibody of the human lgG4 subclass in which S228 is replaced by P and L235 is replaced by E, in that the non-binding of the antibody to the complement factor C1q refers to a measurement by ELISA assay in which the maximum binding (Bmax) of C1q to the antibody at a concentration of 10 pg / ml of the antibody is <30% of the Bmax of the antibody LC 1004-002 of the hu-Mab <P-selectin> cell line LC 1004-002 (DSM ACC2641). In another embodiment, the maximum binding is <20% of the Bmax of the LC 1004-002 antibody of the hu-Mab cell line <Pselectin> LC 1004-002 (DSM ACC2641).

In one embodiment, the present invention provides an antibody that binds to P-selectin, does not bind to complement factor C1q, containing an Fc part derived from human origin and being characterized by the fact that said antibody is an antibody of the subclass lgG1 human, containing at least one mutation in L234, L235, D270, N297, E318, K320, K322, P331 and / or P329 or being an antibody of the human lgG4 subclass in which S228 is replaced by P and L235 is replaced by E, in that the antibody binds to P-selectin with a Kd value of less than 10 ' ⁸ M in a BIAcore assay. In another modality, the range of K _D is 10 ¹¹ to 10 ' ⁹ M.

In one embodiment, the present invention provides an antibody that binds to P-selectin, does not bind to complement factor C1q, containing an Fc part derived from human origin and being characterized by the fact that said antibody is an antibody of the subclass lgG1 human, containing at least one mutation in L234, L235, D270, N297, E318, K320, K322, P331 and / or P329 or being an antibody of the subclass lgG4 huma44 in which S228 is replaced by P and L235 is replaced by E, where the antibody binds at least 1000 times more specifically to P-selectin than to E- and / or L-selectin as measured by the EC50 values in an ELISA assay, where P- and E- and / or L- selectin are coated on the microtiter slide. In another embodiment, EC50 values for E- and L-selectin transfectants are above 100 pg / ml.

In one embodiment, the present invention provides an antibody that binds to P-selectin, does not bind to complement factor C1q, containing an Fc part derived from human origin and being characterized by the fact that said antibody is an antibody of the subclass lgG1 human, containing at least one mutation in L234, L235, D270, N297, E318, K320, K322, P331 and / or P329 or being an antibody of the human lgG4 subclass in which S228 is replaced by P and L235 is replaced by E, in that the antibody inhibits the adhesion of leukocyte-like HL60 cells to the purified Pselectin with a 1C50 value of no more than 1 pg / ml. In another modality, the IC50 value is in the range of 0.08 to 0.5 pg / ml. In yet another modality, the IC50 value is in the range of 0.08 to 0.11 pg / ml.

In one embodiment, the present invention provides an antibody that binds to P-selectin, does not bind to complement factor C1q, containing an Fc part derived from human origin and being characterized by the fact that said antibody is an antibody of the subclass lgG1 human, containing at least one mutation in L234, L235, D270, N297, E318, K320, K322, P331 and / or P329 or being an antibody of the human lgG4 subclass in which S228 is replaced by P and L235 is replaced by E, in what:

(a) adhesion of leukocyte-like HL60 cells to activated platelets is inhibited with an IC50 value of 0.05 to 0.3 pg / ml;

(b) the antibody inhibits the interaction of leukocytes with a platelet monolayer by more than 70%;

(c) the antibody inhibits leukocyte adhesion to activated endothelial cells in a human flow system in the range of 60 to 90% at a concentration of 3 pg / ml;

(d) the antibody does not bind to the C3 protein;

⁴⁵ :? : · · * ·. ::?

(e) the antibody does not stimulate complementary dependent cytotoxicity (CDC);

(f) the antibody does not bind to Fcy receptors on NK effector cells; or (g) the antibody does not stimulate anti-body dependent cell cytotoxicity (ADCC).

In one embodiment, the present invention provides an antibody that binds to P-selectin characterized by the fact that the amino acid sequence of the CDR3 variable heavy chain of said antibody is selected from the group consisting of the CDR3 heavy chain sequences SEQ ID NO: 38, 39, 40, 41 or 42.

In one embodiment, the present invention provides an antibody that binds to P-selectin comprising a variable heavy chain and a variable light chain characterized in that the variable heavy chain comprises the CDR sequences CDR1, CDR2 and CDR3 and CDR1 is selected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, CDR2 is selected from the group consisting of SEQ ID NOs: 33, 34, 35, 36, 37, CDR3 is selected from the group consisting of SEQ ID NOs: 38, 39, 40, 41, 42, wherein said CDRs are selected independently of each other.

In one embodiment, the present invention provides an antibody characterized by the fact that the variable light chain comprises the CDR sequences CDR1, CDR2 and CDR3 and CDR1 is selected from SEQ ID NOs: 43, 44, CDR2 is selected from SEQ ID NOs: 45, 46 and CDR3 is selected from SEQ ID NOs: 47, 48, 49, 50, 51, 52, wherein said CDRs are selected independently of each other.

In one embodiment, the present invention provides an antibody characterized by the fact that said antibody binds to P-selectin and the antibody comprises a variable region independently selected from the group consisting of:

a) the light chain variable domain defined by the amino acid sequence SEQ ID NO: 1 and the heavy chain variable domain defined by SEQ ID NO: 2;

b) the light chain variable domain defined by the amino acid sequence SEQ ID NO: 3 and the heavy chain variable domain defined by SEQ ID NO: 4;

c) the light chain variable domain defined by the amino acid sequence SEQ ID NO: 5 and the heavy chain variable domain defined by SEQ ID NO: 6;

d) the light chain variable domain defined by the amino acid sequence SEQ ID NO: 7 and the heavy chain variable domain defined by SEQ ID NO: 8;

e) the light chain variable domain defined by the amino acid sequence SEQ ID NO: 9 and the heavy chain variable domain defined by SEQ ID NO: 10;

f) the light chain variable domain defined by the amino acid sequence SEQ ID NO: 11 and the heavy chain variable domain defined by SEQ ID NO: 12;

g) the light chain variable domain defined by the amino acid sequence SEQ ID NO: 13 and the heavy chain variable domain defined by SEQ ID NO: 14;

h) the light chain variable domain defined by the amino acid sequence SEQ ID NO: 15 and the heavy chain variable domain defined by SEQ ID NO: 16;

i) the light chain variable domain defined by the amino acid sequence SEQ ID NO: 17 and the heavy chain variable domain defined by SEQ ID NO: 18;

j) the light chain variable domain defined by the amino acid sequence SEQ ID NO: 19 and the heavy chain variable domain defined by SEQ ID NO: 20; and

k) the light chain variable domain defined by the amino acid sequence SEQ ID NO: 21 and the heavy chain variable domain defined by SEQ ID NO: 22

In one embodiment, the present invention provides an anti5?

body that binds to P-selectin, does not bind to complement factor C1q, containing an Fc part derived from Fc of human origin and being characterized by the fact that said antibody is an antibody of the human lgG1 subclass containing at least one mutation in L234, L235, D270, N297, E318, K320, K322, P331 and / or P329 or be an antibody of the human lgG4 subclass in which S228 is replaced by P and L235 is replaced by E, where the antibody comprises the CDR1 regions, CDR2 and CDR3 of the light chain variable domain defined by the amino acid sequence SEQ ID NO: 3 and the CDR1, CDR2 and CDR3 regions of the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 4. In another embodiment, the antibody comprises the light chain variable domain defined by the amino acid sequence SEQ ID NO: 3 and the heavy chain variable domain defined by SEQ ID NO: 4.

In one embodiment, the present invention provides an antibody that binds to P-selectin, does not bind to complement factor C1q, containing an Fc part derived from human origin and being characterized by the fact that said antibody is an antibody of the subclass lgG1 protein containing at least one mutation in L234, L235, D270, N297, E318, K320, K322, P331 and / or P329 or being an antibody of the human lgG4 subclass, where S228 is replaced by P and L235 is replaced by E, in what:

(a) the antibody comprises at least one amino acid mutation in the Fc part causing non-binding to complement factor C1q;

(b) the human heavy chain constant region comprises the amino acid sequence independently selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26 and 28;

(c) the antibody comprises the κ-light chain constant region as defined by SEQ ID NO: 23;

(d) the antibody comprises at least one amino acid mutation that causes non-binding to the C1q complement;

(e) the antibody comprises a heavy chain constant region selected from the group consisting of lgG1v1, lgG1v2 and lgG4v1; or (f) the antibody is a Fab, F (ab ') 2 or chain-like fragment.

In one embodiment, the present invention provides an anti-P-selectin antibody characterized by the fact that a) it is a human or humanized antibody and b) it binds at least 1000 times more specifically to P-selectin than to E- or L- selectin, as measured by EC50 values in an ELISA assay, in which P- and E- and / or L-selectin are coated on the microtiter slide. In another embodiment, the antibody comprises the sequence of amino acids as defined by the heavy chain constant region γ1 in SEQ ID NOs: 24, 25 or 26 or the heavy chain constant region γ4 in SEQ ID NOs: 27 or 28. In yet another In this embodiment, the antibody is produced by a cell line selected from the group consisting of hu-Mab <P-selectin> LC 1004-001 (DSM ACC2640), hu-Mab <P-selectin> LC 1004-002 (DSM ACC2641) and hu + Mab <P-selectin> LC 1004-017 (DSM ACC2642).

It should be understood that the invention provides modalities with definitions as described in paragraphs [00129] to [00141] and also combinations thereof.

The following examples, references, sequence listing and figures are provided to assist in understanding the present invention, the true scope of which is presented in the attached claims. It should _be understood that modifications can be made to the procedures presented without departing from the spirit of the invention.

String Listing Description

SEQ ID NO: 1 LC1004-001 light chain, variable domain of HuMab 1004-001 SEQ ID NO: 2 LC1004-001 heavy chain, variable domain HuMab 1004-001 level SEQ ID NO: 3 LC 1004-002 light chain, variable domain from HuMab 002 SEQ ID NO: 4 LC 1004-002 heavy chain, variable domain HuMab 002 level SEQ ID NO: 5 LC 1004-003 light chain, variable domain

SEQ ID NO: 6 from HuMab 003 LC 1004-003 heavy chain, variable domain SEQ ID NO: 7 HuMab 003 level LC 1004-004 light chain (I), variable domain 5 HuMab 004 (I) SEQ ID NO: 8 LC 1004-004 heavy chain (I), domain SEQ ID NO: 9 HuMab variable 004 (I) LC 1004-004 light chain (II), variable domain 10 SEQ ID NO: 10 HuMab 004 (H) LC 1004-004 heavy chain (II), domain SEQ ID NO: 11 HuMab variable 004 (II) light chain, variable domain of HuMab 005 SEQIDNO: 12 heavy chain, HuMab variable domain 15 SEQ ID NO: 13 005 light chain, variable domain of HuMab 010 SEQ ID NO: 14 (D heavy chain, HuMab variable domain SEQ1DNO: 15 010 (1) light chain, variable domain of HuMab 010 20 (II) SEQ ID NO: 16 heavy chain, HuMab variable domain SEQ ID NO: 17 010 (II) light chain, variable domain of HuMab 010 25 SEQ ID NO: 18 (UI) heavy chain, HuMab variable domain SEQ ID NO: 19 010 (111) light chain, variable domain of HuMab 011 SEQ ID NO: 20 heavy chain, HuMab variable domain 30 SEQ ID NO: 21 011 light chain, variable domain of HuMab 017 SEQ ID NO: 22 heavy chain, HuMab variable domain 017

Qo

⁵⁰ SEQ ID NO: 23 light chain constant region κ SEQ ID NO: 24 γ1 heavy chain constant region SEQ ID NO: 25 heavy chain constant region γ1 of PVA236 / GLPSS331 (IgG 1 v1) SEQ ID NO: 26 heavy chain constant region γ1 of L234A / L235A (lgG1v2) SEQ ID NO: 27 γ4 heavy chain constant region SEQ ID NO: 28 γ4 heavy chain constant region of S228 / L235E (lgG4v1) SEQ ID NO: 29-32 heavy chain CDR1 SEQ ID NO: 33-37 heavy chain CDR2 SEQ ID NO: 38-42 heavy chain CDR3 SEQ ID NO: 43-44 light chain CDR1 SEQ ID NO: 45-46 CDR2 light chain SEQ ID NO: 47-52 Abbreviations: CDR3 light chain

Amino acids are abbreviated with the three letter code (Leu) or a letter (L).

The HuMab 00X antibody is also called the 00X antibody.

L234 means the amino acid leucine at position 234 according to the North American numbering (Kabat).

L234A means that the amino acid leucine at position 234 is replaced by alanine.

PVA236 means that in the ELLG 236 region of lgG1 or EFLG of lgG4 it is rectified in PVA.

GLPSS331 means that the 331 ALPAP region of lgG1 or GLPAP of lgG2 is switched to GLPSS.

Rectifications in other IgG subclasses similarly.

EXAMPLES

Generation of a hybridoma cell line that produces anti-P-selectin antibodies

Hybridoma culture

HuMab hybridomas were grown in IMDM (Cambrex), Bovine Fetal serum clone 1 (Perbio Science), cloning factor of Hybridoma of origin (Igen), sodium pyruvate, penicillin / streptomycin, 2mercaptoethanol, HAT (Sigma-Aldrich) and Kanamycin (Invitrogen) at 37 ° C and 5% 5 CO ₂ .

Generation of a hybridoma cell line that produces anti-P-selectin antibodies

Immunization procedure of transgenic mice

Protocol A:

10 transgenic HCo7 mice (5 males and 5 females), strain GG2201 (Medarex, San José, CA, USA) were immunized with a recombinant truncated form of P-selectin which lacks the transmembrane and cytoplasmic domain of P-selectin and which was acquired from R&D Systems. For the first immunization, 50 µg of recombinant P-selectin, dissolved in 100 µl of PBS, was mixed with 100 µl of complete Freund's adjuvant. For the remaining immunizations, recombinant P-selectin coupled to KLH was used. For the second immunization, 50 µg of recombinant KLH-coupled P-selectin was dissolved in 100 µl of PBS and mixed with 100 µl of Freund's incomplete adjuvant. For the remaining immunizations, 20 µg of recombinant KLH-coupled P-selectin was dissolved in 100 µL of PBS and mixed with 100 µl of Freund's incomplete adjuvant. Immunizations were administered alternating interperitoneally and subcutaneously, starting with an interperitoneal immunization.

Protocol B:

3 transgenic HCo7 mice (all female) and 3 KM (all male), strain GG2489 (Medarex, San José, CA, USA) were immunized with obsolete human platelet purified full-length P-selectin via immunoaffinity chromatography (see below ). For the first immunization, 50 pg of purified P-selectin dissolved in 100 μΙ of

PBS were mixed with 100 µl of Freunds complete adjuvant (CFA; Difco Laboratories, Detroit, USA). For the second immunization, 50 pg of the purified Pselectin dissolved in 100 pl of PBS was mixed with 100

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μΙ of Freunds incomplete adjuvant (ICFA; Difco).

For all other immunizations, 20 pg of purified P-selectin was used and mixed with 100 μί of Freund's incomplete adjuvant.

Antigen-specific ELISA

Anti-P-selectin titrations in the serum of immunized mice were determined by an antigen-specific ELISA. The slide (96-well flat bottom ELISA slide, Greiner) was coated with 0.1 pg / ml of purified P-selectin dissolved in PBS and coated overnight at room temperature. Thereafter, the wells were blocked with PBSTC (PBS containing 0.05% Tween 20 (Sigma-Aldrich Chemie BV) and 2% chicken serum (Gibco)) for 1 hour at room temperature.

Serum samples tested were diluted 1: 100 in PBSTC and added to the wells. Serum obtained from mice before immunization was dissolved at 1: 100 in PBSTC and used as a negative control. A mouse antibody directed against human P-selectin (1/7, produced in the laboratory by Roche Basel) was dissolved 1: 100 in PBSTC and used as a positive control. The slides were incubated for 1 hour at room temperature. Subsequently, the slides were washed twice using PBST (PBS containing 0.05% Tween 20). Gt-ahulgG-HRP (Jackson) was diluted 1: 5000 in PBSTC and added to the wells containing the tested samples and the negative control. Rb-a-mlgG (Jackson) was diluted 1: 3000 in PBSTC and added to the wells containing the positive control. The slides were incubated for 1 hour at room temperature. Finally, the slides were washed twice using PBST and developed with freshly prepared ABTS® solution (1 mg / ml) (ABTS: 2,2'-azino bis (3-ethylbenzthiazoline-6-sulfonic acid) for 30 minutes at room temperature (TA) in the dark, absorbance was measured at 405 nm.

Reinforcement of mice

When the anti-P-selectin titrations in the serum were sufficient, the mice additionally received a boost twice with 20 µg of recombinant human P-selectin in 100 µL of PBS, intravenously 4 and 3 days before fusion.

Hybridoma Generation

The mice were sacrificed and the spleen and lymph nodes that flank the abdominal aorta and vena cava were collected. The fusion of splenocytes and lymph node cells with the SP 2.0 cell fusion partner was performed according to standard operating procedures.

Human monoclonal antibodies with heavy and light sequences of SEQ ID NOs 1-22 were obtained through the immunization procedure.

k-ELISA

To determine whether the hybridomas that resulted from the fusion generate human antibodies, an κ-ELlSA was performed. ELISA slides were coated with rat anti-human IgG κ-light chain antibody (DAKO) diluted 1/10000 in PBS by overnight incubation at 4 ° C. After discarding the wells, the slides were blocked by incubating with PBSTC for 1 hour at room temperature. After that, the wells were incubated with hybridoma culture supernatant, diluted 1/2 in PBSTC. Culture medium dissolved at 1/2 in PBSTC was used as a negative control, κ-mild positive mouse serum diluted to 1/100 in PBSTC served as a positive control. Subsequently, the wells were washed twice and incubated with HRP-conjugated human anti-IgG F (ab ') 2 (DAKO), diluted to 1/2000 in PBSTC for 1 h at 37 ° C. The wells were washed twice and the assays were developed with freshly prepared ABTS® solution (1 mg / ml) for 30 minutes at room temperature (RT) in the dark. Absorbance was measured at 405 nm in an ELISA slide reader. Cloning and sequence analysis of HuMab anti-P-selectin variable domains (κ-light and γ1-heavy chains)

The nucleotide sequences encoding the V _L light chain variable region and the HuMabs P-selectin V _H heavy chain variable region were isolated using a standard cDNA / PCR synthesis procedure.

Total RNA was prepared from 1x10 ⁶ - 1x10 ⁷ hybridoma cells using the RNeasy ® Mini Kit (Qiagen). Hybridoma-derived RNA was used as a model for the 1 ^- strand cDNA synthesis, which was performed according to a conventional method that makes use of an oligo dT primer. 2 ^- strand cDNA synthesis and additional PCR amplification of the cDNA encoding fragments of cDNA encoding V _L and V _H was performed with light and heavy chain reverse primers complementary to the nucleotide sequences of the κ-light chain constant region and γ1-heavy and 5'-specific heavy and light chain primers, respectively. The PCR products were cloned using the TOPO TA cloning kit from Invitrogen ™ Life Technologies and pCR4-TOPO as a cloning vector. The cloned PCR products were identified by restriction mapping of the appropriate plasmids using EcoRI for digestion and expected / calculated DNA fragment sizes of about 740 and 790 bp for Vl and Vh, respectively.

The DNA sequence of the cloned PCR fragments was determined using double strand sequencing.

The software package GCG (Genetics Computer Group, Madison, Wisconsin) version 10.2 was used for general data processing. The DNA and protein sequences were aligned using the CLUSTALW GCG module. The sequence alignments were placed in tables, edited and color coded using the GENEDOC program (version 2.1). Construction of expression plasmids for HuMab anti-P-selectin lgG1

The genes encoding the HuMab anti-Pselectin light and heavy chains were separately assembled into mammalian cell expression vectors.

In this way, the gene segments encoding the HuMab anti-P-selectin light chain variable region (Vl) and the human κ-light chain constant region (Cl) were joined together, as well as gene segments for the region HuMab anti-P-selectin heavy chain variable (V _H ) and the human γ1-heavy chain constant region (C _H rCh2Ch3 hinge) General information regarding the human light and heavy chain nucleotide sequences from which the codon usage can be deducted is provided at: Kabat, EA, Wu, TT, Perry, Η. M., Gottesman, KS and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No. 91-3242.

The HuMab anti-P-selectin κ-light chain transcription unit is composed of the following elements:

• The immediate early enhancer and human cytomegalovirus (HCMV) promoter, • A synthetic 5-UT, including a Kozak sequence, • A murine immunoglobulin heavy chain signal sequence, including the signal sequence intron, • The cDNA of cloned HuMab anti-P-selectin variable light chain arranged with a single Bsm \ restriction site at the 5 'end and a donor binding site and a single Λ / ofl restriction site at the 3' end, • The constant region of the human genomic κ-gene, including intron 2 of the mouse lg-κ intensifier [Picard, D., and Schaffner, W. (1984) Nature 307, 80-82] and • The κ-polyadenylation signal sequence ( poly A) human immunoglobulin.

The HuMab anti-Pselectin γ1-heavy chain transcription unit is composed of the following elements:

• The immediate early enhancer and human cytomegalovirus (HCMV) promoter, • A synthetic 5'-UT, including a Kozak sequence, • A modified murine immunoglobulin heavy chain signal sequence, including the intron of the signal sequence, • · »····· · * • The cloned HuMab anti-P-selectin variable heavy chain cDNA cloned with a single Bsmi restriction site at the 5 'end and a donor binding site and a single restriction site Λ / οίΙ at the 3 'end, · The constant region of the human genomic γ1-heavy gene, including the mouse Ig μ-enhancer [Neuberger, MS (1983) EmboJ2, 1373-1378], • The polyadenylation sequence (poly A) human γΐ-immunoglobulin.

Functional elements of HuMab anti-P-selectin kleve and γ1-heavy chain expression plasmids: in addition to the HuMab κ-light and γ1-heavy chain expression cassette, these plasmids contain :

• A hygromycin resistance gene · An origin of replication, oriP, of the Epstein-Barr virus (EBV) • An origin of replication of the pUC18 vector, which allows replication of this plasmid in E. coli and • A β gene -lactamase which gives resistance to ampicillin in E. coli.

Construction of HuMab anti-P-selectin IgG4 expression plasmids

An anti-P-selectin y4-heavy chain prototype expression plasmid was derived from the anti-P-selectin γ1-heavy chain expression plasmid replacing the human genomic γ1-constant region and the y1 polyadenylation (poly A) signal sequence -immunoglobulin by the human genomic y4-constant region and the y4-immunoglobulin polyadenylation signal sequence.

For the expression of HuMab anti-P-selectin κ-light chains, the same expression plasmids were used as described 30 for IgG1 (see above).

Construction of expression plasmids for anti-P-selectin mutants (variants) αί lqG1 and lqG4

Expression plasmids encoding the mutant anti-P-selectin γ1- and γ4 chains were created by site-directed mutagenesis of the wild-type expression plasmids using the QuickChange ™ Site-Directed Mutagenesis Kit (Stratagene).

The following mutants were generated for LC1004-002. Amino acids are numbered according to North American numbering [Edelman, G. M., Cunningham, B. A., Gall, W. E., Gottlieb, P. D., Rutishauser, U. and Waxdal, M. J. (1969) Proc Natl Acad Sei USA63, 78-85; Kabat, E. A., Wu, T. T., Perry, Η. M., Gottesman, K. S. and Foeller, C. (1991) Sequences 10 of Proteins of Immunological Interest, Fifth Ed., NIH Publication No. 913242].

TABLE 1 Isotype Abbreviation Mutations description igGl lgG1v1 PVA-236; GLPSS331 As specified by E233P; L234V; L235A; delta G236; A327G; A330S; P331S SEQ ID NO: 25 The GIU23sLeu234l-eu235Gly236 amino acid sequence of the human γ1-heavy chain is replaced by the amino acid sequence Pro233Val234Ala235 of the human y2-heavy chain. The Ala327Leu328Pro329Ala33oPro33i amino acid sequence of the human γ1-heavy chain is replaced by the amino acid sequence Gly327Leu328Pro329Ser33oSer33i of the human y4-heavy chain igGl lgG1v2 L234A; L235A SEQ ID NO: 26 The Leu234Leu ₂ 35 amino acid sequence of the human γ1 chain is replaced by the Ala234Ala235 amino acid sequence lgG4 lgG4v1 S228P; L235E SEQ ID NO: 28 Ser228 of the human y4-heavy chain is replaced with Pro ₂ 28 and Leu ₂ 35 of the human y4-heavy chain is replaced with GIU235

Production of recombinant anti-P-selectin HuMabs

Recombinant HuMabs were generated by transient transfection of adherent K293-EBNA cells (ATTC No. CRL-10852) cultured in DMEM (Gibco) supplemented with FCS with ultra low content of 10% IgG (Gibco), 2 mM Glutamine (Gibco) , 1% v / v non-essential amino acids (Gibco) and 250 pg / ml G418 (Roche). For transfection, Fugene ™ 6 (Roche) Transfection Reagent was used in a ratio of reagent (pl) to DNA (pg) ranging from 3: 1 to 6: 1. Immunoglobulin light and heavy chains were expressed from two different plasmids using a 1: 2 to 2: 1 light chain to plasmid encoding heavy chain encoding plasmid. HuMab containing cell culture supernatants was collected on days 4 to 11 after transfection. Supernatants were stored at -20 ° C until purification.

General information regarding recombinant expression of human antibody, for example, in HEK293, is provided in: Meissner, P., Pick, H., Kulangara, A., Chatellard, P., Friedrich, K. and Wurm, FM ( 2001) Biotechnol Bioeng 75, 197-203.

Determination of the affinity of anti-P-selectin HuMabs

Equipment:

Instrument: BIACORE® 2000

Sliver: CM5

Coupling: coupling with amine

Buffer: HBS (HEPES, NaCI), pH 7.4, 25 ° C

For affinity measurements, rabbit anti-human Fcy antibodies (Dianova) were coupled through amine coupling to the chip surface for presentation of the antibody against P-selectin. Approximately 400 RU of anti-P-selectin antibodies were bound. Recombinant p-selectin (R&D Systems) was added in concentrations ranging from 0-50 nM. The association was measured by injection of P-selectin for 120 seconds; dissociation was measured by washing the chip surface with buffer for 180 seconds. The affinity data for different antibodies to P-selectin are shown in Table 2. Using Soft ware Biaevaluation, the kinetic data were adapted for a 1: 1 model of Langmuir P-selectin binding to the monoclonal antibody shown.

TABLE 2

Affinity data measured by SPR (BIACORE® 2000)

HuMab Antibody ka (1 / Ms) kd (1 / s) KA (1 / M) KD (M) 001 6.08 x 10® 4.19 x 10 ' ⁴ 1.45 x 10 ⁹ 6.89 x 10 ' ¹ ° 002 8.10 x10 ⁵ 2.13 x 10 ' ³ 3.81 x 10 ⁹ 2.63x10 ' ⁹ 003 6.60 x 10® 2.91 x 10 ⁴ 2.27 x 10 ⁹ 4.41 x 10 ^'10 005 8.42 x 10 ⁵ 2.89 x 10 ' ⁴ 2.91 x 10 ⁹ 3.43 x 10 ^'10 011 1.77x10® 2.38x10 ' ³ 7.44 x 10® 1.34 x 10 ' ⁹ 012 1.08x10® 1.25 x 10 ' ⁴ 8.65 x 10 ⁹ 1.16 x 10 ' ¹ ° 013 1.46x10® 2.02x10 ' ⁴ 7.22 x 10 ⁹ 1.39 x 10 ^'10 017 7.79x10® 1.39 x 10 ' ⁵ 5.59x10® 1.79 x 10 ^'11

Inhibitory activity of antibodies to P-selectin in a cell-based rosetting and adhesion assay

Materials and methods:

Cell adhesion assay: In the cell adhesion assay, the effect of HuMabs on the adhesion of leukocyte-like HL60 cells (ATCC CCL 240) to P-selectin coated on microtiter slides was evaluated. HL60 cells were labeled with BCECF-AM (2 ', 7'-bis- (2-carboxyethyl) -5- (e-6) -carboxyfluorescein acetoxymethyl ester; Cat. No 216254, Calbiochem). P-selectin purified full length (purification procedure, see above) at a concentration of 1 ug / ml in buffer containing NaCl 150 mM, CaCl _2-1 mM MgCl _2-1 mM, 20 mM Tris (pH 7.4) plus 0.0005% Tx100 was coated overnight at 4 ° C on 96 well slides (Nunc Immunoplate Maxisorp F96). Thereafter, the wells were blocked with the aforementioned buffer containing 3.5% bovine serum albumin (BSA, Fluka) for 2 h at room temperature (RT). The wells were pre-incubated with 50 μΙ of different dilutions of the HuMabs to P-selectin or antibodies to the reference mouse P-selectin (WAPS 12.2, respective hybridoma cell line provided by ATCC) in the above mentioned buffer containing BSA a 1% for 20 minutes in TA. HL60 labeled cells (50 μΙ, 70,000 cells / well) were added and allowed to bind for 45 min in RT. In some experiments, HL60 cells were pre-incubated with 20 pg / ml of human IgG1 for 30 minutes before being added to the wells in order to block Fc receptors. After removing the unbound HL60 cells by light washing (4 times with the aforementioned buffer), the adherent cells were subjected to lysis with 120 μΙ of NP-40 (Fluka; 1% in H2O). 100 μΙ of the supernatants were transferred to the slides to measure their fluorescence at an excitation wavelength of 485 nm and an emission of 538 nm using an LS 50B luminescence spectrometer (Perkin Elmer).

Rosette assay: To assess the effect of antibodies on the interaction of activated platelets with HL60 cells, a rosette assay (Jungi et al., Blood 67: 629 (1986)) in combination with double color cytofluorimetric analysis (Evangelista et al., Blood 88: 4183 (1996) was applied. Washed human platelets were prepared as described (Fox et al., Methods Enzymol 215: 45 (1992)). They were activated with thrombin (final conc. 1 U / ml) for 5 min and labeled with a FITC-conjugated human anti-GPIIb pl-36 antibody (Kouns et al., J Biol Chem 267: 18844 (1992)). Then, 1.4-2x10 ⁶ platelets within 70 μΙ of tyrode solution were incubated with different dilutions of HuMabs (100 μΐ) in the dark for 30 min in RT. 50 μΙ of HL60 cell suspension (in Tyrode solution) adjusted to 20x10 ⁶ / ml was added. HL60 cells were labeled by incubation with 20 μΙ of Anti-human CD45 Ab PE (phycoerythrin) -conjugate (Code N 555483, Pharmingen) After incubating HL60 cells labeled with platelets and HuMabs for 30 min at room temperature in FACS tubes (Becton Dickinson), the formation of mixed aggregates or rosettes was analyzed by measuring the fluorescence marker platelets and HL60 cells using a FACScan (Becton Dickinson). Front and side scatter signals, as well as green (FITC) and red (PE) were acquired through Yoga7 /

J * φ φ φ · · · · · · · rhythmic with an excitation wavelength of 488 nm and an emission wavelength of 530 nm (FITC) and 570 nm (PE), respectively. Electronic compensation was used to remove spectral overlap. HL60 cells were identified based on front and side dispersion. Triggering of events identified as HL60 cells was performed to exclude simple platelets. Five hundred events triggered by HL60 cells were measured for each sample. A sample in which platelets not activated or activated by thrombin were mixed with HL60 cells in the presence of EDTA (10 mmol / l) was used to adjust a threshold on the green fluorescence scale. The percentage of HL60 cells above the threshold represents the percentage of HL60 cells bound to platelets. The deviation of fluorescence from the platelet marker to lower fluorescence values reflects the reduction in the number of mixed aggregates, with a greater number of adherent platelets in favor of an increase in the number of aggregates mixed with a low number of adherent platelets. Results:

In the HL60 cell adhesion assay, antibodies to Pselectin inhibited the adhesion of HL60 cells to purified P-selectin with IC 50 values in the range of 0.08 - 0.5 pg / ml. Although the mutations were introduced into the Fc portion of the antibody, the HuMabs lgG4 and lgG1 variants were more potent than the original antibody with IC 50 values of 0.08 - 0.11 pg / ml, as shown in Figurei. When preincubating HL60 cells with human IgG1, the potency of the original antibodies without mutation is also increased, with a reduction of around 3 to 4 times in the IC50 value, as shown for HuMab 002 in Figurei. This finding suggests that the increased effectiveness of the mutants in the adhesion assay is primarily due to the elimination of adhesion of HL60 cells to P-selectin via the Fc portion of the antibody to Fcy receptors.

In the rosette assay that assesses the adhesion of human platelets activated by expressing P-selectin to HL60 cells, the IC50 values of HuMabs were well below those of the adhesion assay due to the lower number of P-selectin receptors in that assay (IC50: 0.05 0.3 pg / ml, preferably between 0.05 and 0.2 pg / ml). The effectiveness of the Fc variants of the respective HuMabs tends to be increased when compared to the original antibody without mutation (Figure 2). Pre-incubation of HL60 cells with lgG1 and lgG4 before incubation with activated platelets does not significantly affect the inhibitory activity of the original mutants and antibodies, indicating a less pronounced role of Fcy-mediated receptor binding in the rosetting assay when compared to the adhesion assay .

Cross-reactivity of antibodies to P-selectin with P-selectin from animal species

Materials and Methods: The cross-reactivity of HuMabs to Pselectin was assessed by measuring (i) the binding of HuMabs to activated platelets of rat and cynomologus monkeys using FACS analysis and (ii) their inhibitory activity in the rosette assay that assesses adhesion from rat platelets and cynomologus to HL60 cells.

To measure the binding of HuMabs to activated rat and cynomologus platelets, washed rat and cynomologus platelets were prepared in a similar manner to the preparation of washed human platelets (see above). They were activated with thrombin (final conc. 1 U / ml) for 5 min. The activated platelets were incubated with different dilutions of the HuMabs (20 μΙ) for 30 min in RT. After binding the HuMabs, the platelets were fixed with 2% PFA in RT for 15 min. The samples were washed with Tyrode buffer and resuspended in 300 ml of Tyrode. The binding of the HuMabs was detected with a FITC-rabbit anti-human IgG F (ab ') ₂ fragment (Code No. F0056, Dako). As a control antibody that inhibits rat P-selectin, a rabbit anti-human polyclonal anti-P-selectin antibody (Code No. 09361 A, Pharmingen) was used.

To measure the inhibitory effect of HuMabs on P-selectin in the rosetting assay, washed rat platelets and cynomologus were prepared as described above for human platelets. The rosetting assay was performed essentially as described for human platelets.

.......

φ φ φ φ · * * · # · ·

For cynomologus platelet labeling, the FITC-conjugated human anti-GPIIb pl-36 antibody was used, while the rat platelets were labeled with the conjugated FITC mouse anti-CD61 antibody (Code No. 554952, Pharmingeh).

Results: It has been shown that none of the antibodies of the invention specific to P-selectin which inhibit human-mediated P-selectin functions bind to rat P-selectin or inhibit the formation of mixed aggregates consisting of rat platelets and HL60 cells, as shown for some examples in Figure 3a. However, HuMabs to P-selectin bind and inhibit cynomologus P-selectin (Figure 3b).

Selectivity of antibodies to P-selectin vs E- and L-selectin

Materials and Methods: The selectivity of HuMabs to P-selectin vs E- and L-selectin was determined in a cell-free ELISA that measures the binding of antibodies to recombinant E- and L-selectin (ADP1 and ADP2, R&D Systems) and a cell-based ELISA that measures the binding of antibodies to E-selectin-CHO transfectants and Lselectin-300.19 transfectants (transfectants were generated as described in Goetz et al., J Cell Biol 137: 509 (1997); Ley et al. , Blood 82: 1632 (1993)).

In the cell-free ELISA, recombinant P-, E- or L-selectin at a concentration of 1 pg / ml in buffer containing 150 mM NaCI, 1 mM CaCl _2, 1 mM MgCl ₂ , 20 mM Tris (pH 7.4) plus 0.0005% Tx100 was coated overnight at 4 ° C on 96 well slides (Nunc Immunoplate Maxisorp F96). Thereafter, the wells were blocked with the aforementioned buffer containing 3.5% bovine serum albumin (BSA, Fluka) for 2 h at RT. The wells were pre-incubated with 50 μΙ of different dilutions of the HuMabs to P-selectin or antibody to P-, reference mouse E-selectin (BBA26; R&D Systems) and calf L-selectin antibody (AF728; R&D Systems ) in the above-mentioned buffer containing 1% BSA overnight in RT. The binding of HuMabs was detected using biotinylated human anti-IgG (Amersham, RPN1003, final concentration 1: 1000) or, for control antibodies, the

7 /

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Corresponding anti-mouse IgG or biotinylated antibezer. After 1 h of incubation, the wells were washed (3 times) with the aforementioned buffer and 0.1 ml of biotinylated streptavidin-peroxidase complex (Amersham, RPN1051) diluted 1: 750 in the mentioned buffer containing 0.1 BSA % was added over 30 min. The wells were then washed and 0.2 ml of peroxidase substrate solution containing ABTS (2,2'-azino-di- (3-ethylbenzthiazoline sulfonate, Boehringer, Mannheim) was added (ABTS stock solution: 1 ml of 40 mM ABTS, 5 μΙ of 30% H2O2 and 20 ml of 0.1 M Na-Acetat, 0.05 M NaH ₂ PO ₄ ) The reaction was stopped after about 10 min using 50 μΙ of citrate to 0.1 Me 0.01% NaN ₃ The color reaction was read at 405 nm.

In the cell-based ELISA, P- and E-selectin-CHO transfectants, after releasing the cells with cell dissociation solution (Sigma C5914), they were cultured in each 96-well slide well (TC Microwell F96 Nunc 167008) set to 100,000 cells / well and cultured in the respective media overnight at 37 ° C (medium for P-CHO-transfectants: DMEM + 10% FCS + 2 mM Glutamine + 100 U / ml penicillin / 100 pg / ml streptomycin; medium for E-selectin transfectants: HAM F-12 + 10% FCS + 2 mM Glutamine + 100 U / ml penicillin / 100 pg / ml streptomycin + 0.1% Fungizone + 100 pg / ml Neomycin ). After removal of the media and blocking the wells with TA buffer (150 mM NaCl, mM CaCl _{2-1, 2-1} mM MgCl, 20 mM Tris (pH 7.4)) containing 3% TopBlock (Code No. TB232010; I swear) for 1 h, 50 μΙ of different dilutions of HuMabs to P-selectin or antibody to Pe reference mouse E-selectin (see above) in the above-mentioned buffer containing 1% TopBlock and 0.1% azide were added and incubated for 60 min in RT. After washing the wells (4 times), bound antibodies were detected using the same steps as mentioned above for the cell-free ELISA.

Since L-selectin / 300.19 cells are cells in suspension, the cell-based ELISA format had to be modified by placing L-selectin-300.19 transfectants in 96-well polystyrene filter slide wells (Corning 3510). Using filter slides, blocking and incubation solutions were removed by filtering them through the bottom of the slides, but otherwise the protocol was similar to the one using P- and E-selectin-CHO cells. As controls, CHO and 300.19 non-transfected were used.

Results:

The antibodies of the invention were highly selective vs E- and Lselectin. They bound to P-selectin-CHO cells with EC50 values in the range of 0.01 to 0.08 pg / ml, preferably in the range of 0.01 to 0.04 pg / ml, while EC50 values on E-selectin-CHO cells and Lselectin-300.19 were clearly above 50 pg / ml, preferably above 100 pg / ml. HuMab 002 had the highest selectivity, with a selectivity factor vs E- and L-selectin more than 4,000 times in the cell-based ELISA. In addition, HuMab 002 does not bind to E- and L-selectin transfectants above baseline levels to a concentration of 100 pg / ml. The selectivity of the Fc lgG4v1 and lgG1v1 variants of HuMab 002 is similar to that of the original HuMab 002 (Figures 4a-c).

Ex vivo inhibitory activity of antibodies to P-selectin in a fully human blood flow system

Effect of HuMabs on P-selectin on leukocyte adhesion to a platelet monolayer

Materials and methods:

To address the effect of antibodies to P-selectin on the recruitment of leukocytes to sites of damage to the wall of platelets and thrombi, a human blood flow system that allows the measurement of the interaction of human leukocytes with human platelets at different rates shear was used essentially as described (Kirchhofer et al., Blood 89: 1270 (1997)). In a parallel slide perfusion device, whole human blood extracted from the antecubital vein of a healthy donor was perfused over a collagen surface simulating the wall of an injured naked blood vessel. Collagen coated coverslips were prepared as described (Kirchhofer et al.,

7C

Blood 89: 1270 (1997)). They were positioned in three perfusion chambers with slides in parallel. To allow the measurement of different shear rates (65 / s and 280 / s), different dimensions of reperfusion chambers were used and the blood was perfused over the collagen-coated coverslips in a constant blood flow of 1 ml / min, the which was controlled by individual cylinder pumps positioned distal to the infusion device. Immediately after collecting blood from the vein and separating the blood into three tubes, a GPIIb / llla inhibitor (0.5 pmol / lamifiban) is added to prevent platelet aggregation and generate platelet monolayers. At the same time, antibodies to P-selectin (the HuMabs, mutants, respective reference antibodies or human IgG1 and IgG4 as controls) were administered in different concentrations and the blood-inhibitor mixture was then placed in the perfusion chamber containing the coverslips collagen coated. After a 5.5 minute infusion period, PBS is perfused through the infusion chamber without interrupting the flow for 3 min. After a rapid interruption of the flow, the chambers were fixed with 3% paraformaldehyde in PBS at 1 ml / min for 2 min. Then, the coverslips were removed from the chambers, fixed again for 1 h in 3% paraformaldehyde in PBS at 4 ° C and stored in PBS-0.03% sodium azide. To assess the number of leukocytes that adhered to the platelet monolayer, after air drying, the coverslips were stained with Diff-Quick solution (Dade Behring AG) and embedded in Merckoglas (Merck, Germany). An image analysis system (MCID, Imaging Research Inc.) was used to determine the number of leukocytes that adhered to a standard area oriented perpendicular to the blood flow 1 mm from the beginning of the coverslip. At a shear rate of 65 / s and 280 / s, the area over which the number of leukocytes was counted comprised 3.1 mm and 2.1 mm, respectively.

Results:

HuMabs to P-selectin inhibited leukocyte adhesion to the platelet monolayer in a concentration-dependent manner. At a shear rate of 65 / s and a concentration of 10 pg / ml, Hu

..........

Mabs inhibited leukocyte adhesion by 60 - 99%, preferably 70-99%. The inhibitory effect of HuMabs was more pronounced at the higher shear rate of 280 / s (closer to the arterial situation) when compared to the venous shear rate of 65 / s. In general, at a stripping rate of 280 / s, the number of leukocytes that adhered was less than 65 / s. When comparing the Fc variants with the respective original antibodies, they had similar inhibitory activity in the ex vivo perfusion chamber, as demonstrated for HuMab 002 and its lgG4v1 and lgG1v1 variants (Figure 5). The increased inhibitory activity of the mutants vs the original antibody found in the in vitro assays was not observed in the ex vivo perfusion chamber, which may be due to the saturation of leukocyte Fcy receptors in intact human blood.

Effect of HuMabs on P-selectin on leukocyte adhesion to endothelial cells

Materials and methods:

To address the HuMabs' anti-inflammatory potential to Pselectin under shear conditions, the aforementioned human blood flow system was used in a setting in which endothelial cells were coated over the coverslips. Human umbilical vein endothelial cells (HUVEC) from umbilical cords were isolated by digestion with Type II collagenase (Roche Switzerland) according to the method of Jaffe et al., 1993 (Please add full quote). They were grown in tissue culture flasks coated with 1% gelatin in 199 medium (M199, Sigma, Germany) supplemented with 20% fetal goat serum (Gibco, Auckland), 100 IU / ml penicillin (Gibco, Auckland) , 0.1 mg / ml streptomycin (Gibco, Auckland), 2 mmol / l L-glutamine (Gibco, Auckland), 10 U / ml heparin (Sigma) and 50 pg / ml EC growth supplement ( Sigma, Germany). HUVECs were grown until confluence (approximately 4 days), undergoing passes with trypsin / ethylendiaminatetraacetic acid (Gibco, Auckland) and grown on Thermanox plastic coverslips (approximately 200,000 ECs / coverslip) previously coated with 1% gelatin (Fluka, Germany). HUVECs were left to

_.. ....... is seated became confluent over 1-2 days. They were stimulated with 20 ng / ml of IL-4 (R&D Systems) 24 h before the start of the infusion and with 10 ' ⁴ M histamine (Fluka, Germany) 5-10 min before the infusion. Each experiment was performed with HUVECs in pass 1. The coverslips with stimulated HUVECs confluent monolayers were positioned in the perfusion chambers with slides in parallel as described above. Similar to the perfusion experiments described above, healthy blood was collected from healthy donors. However, in these experiments, the blood was anticoagulated with a thrombin inhibitor Ro-46-6240 (10 μΜ) and preincubated with different concentrations of antibodies to P-selectin (HuMabs, mutants, respective reference antibodies) or human IgG1 and IgG4 as controls for 5 min just before the infusion on activated endothelial cells. The blood flow was adjusted to 1 ml / min, the shear rate to 65 / s and the perfusion time to 5.5 min. After a 3 min wash period with PBS, the HUVECs with adherent leukocytes were fixed with 3% paraformaldehyde for 2 min under the same flow conditions as described. Then, the coverslips were removed from the chambers, immersed in a fresh fixative for 1 h and stored in PBS-0.02% sodium azide. For morphometric analysis, the leukocytes were stained with a mouse antibody against the common CD45 leukocyte antigen, which was previously labeled using a modified biotinylated anti-mouse immunoglobulin (Animal Research Kit, Dako, USA). The nuclei were counterstained with hematoxylin (JT Baker, Holland).

Results:

The stimulation of HUVECs with the combination of IL-4 and histamine resulted in the expression of P-selectin and the adhesion of different types of leukocytes with granulocytes (including PMNs and eosinophils) that constitute the prevailing portion of the leukocytes that adhere. The HuMabs of the invention inhibited the adhesion of the total leukocyte population by 60-90% at 3 μg / ml. In general, the inhibitory activity of Fc variants was not significantly different from that of HuMabs without mutation.

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HuMabs to P-selectin demonstrate a differential effect on different types of leukocytes. The effect on granulocytes is more pronounced when compared to mononuclear leukocytes. The antibodies according to the invention inhibited the adhesion of granulocytes (including PMNs and eosinophils) by 90-99%, monocytes by 50-88% and lymphocytes by 5-40%. The respective decrease in the absolute numbers of the different types of leukocytes is representatively provided for lgG4v1 in Figure 6. Potential of HuMabs to P-selectin to activate the complement system ELISA binding to C1q and C3c:

To determine the ability of the antibodies of the invention to induce C1q binding and C3 activation, an ELISA approach was used. C1 q is part of the adaptive immune system and, when bound to immune complexes, triggers the sequential activation of various zymogens. Enzymes, in turn, cause the dividing of C3 molecules, which can result in the onset of inflammatory reactions, opsonization of foreign or abnormal particles and lysis of cell membranes.

In principle, the ELISA slide is coated with antibody concentration ranges, to which human C1 q or pooled human serum, as a source of C3, is added. Binding to C1q or Ο3ε is detected by an antibody directed against human C1q or Ο3ε, followed by a peroxidase-labeled conjugate.

HuMab 002 (the hybridoma - and material derived from transient transfectoma, its mutated variants and control antibodies were tested at concentrations of 0.16-20 pg / mL As a negative control, a human IgG4 (CLB, Netherlands, 0.5 pg / ml of stock), which binds to C1q very weakly, was used.Human lgG1 (Sigma, 2 μg / ml of stock) was incorporated as a positive control.To detect C1q, a rabbit antibody directed against C1q (Dako) and a swine rabbit anti-IgG antibody, conjugated to horseradish peroxidase (Sigma), was used to detect 63ε, a mouse anti-human C3 antibody and a mouse anti-IgG antibody of rabbit, conjugated with horseradish peroxidase (Sigma) were applied.

The calculations regarding the EC50 values or maximum binding at 10 pg / ml (Bmax) of the tested HuMab were determined using non-linear regression curve adaptation (binding to a site) using the Graphpad Prism software.

Results:

HuMab 002 according to the invention was able to bind to C1q effectively, as indicated by EC50 values of 0.946 gg / ml and 1.159 gg / ml and Bmax values (OD405) of 0.987 and 0.711 for the hybridoma and derived material transfectome, respectively. As expected, 10 the human IgG4 negative control does not bind to C1q, as indicated by a Bmax value of 0.222 to OD405. However, all three tested Fc variants (lgG4v1, lgG1v1, lgG1v2) had lost their ability to bind to C1q, as shown by OD405 Bmax values of 0.132, 0.119 and 0.132, respectively (Table 3). In accordance with C1q-binding capacities, deposition of C3 in HuMab 002 (hybridoma - and transfectoma-derivative) occurred in an anti-drug dependent manner and EC50 values ranged between 2.7 pg / ml and 8.3 pg / ml. However, all three Fc variants were unable to initiate C3 deposition, as indicated by B40 values in OD405 of 0.104, 0.156 and 20 0.133, respectively (Table 3).

Since HuMab 002 interacts with complement components, this antibody has the intrinsic potential to induce CDC in vivo. Therefore, the Fc part of that antibody is modified according to the invention.

TABLE 3 C1q ELISA C3 ELISA Bmax (OD405 to 10 gg / ml) Base (OD405) Bmax (OD405 at 10 gg / ml) Base (OD4Q5) HuMab 002 (hybridoma) 0.987 0.079 4.47 0.098 lgG4v1 0.132 0.104 lgG1v1 0.119 0.156

---------------------------------------- ·, - · · · ·  TABLE 3 C1q ELISA C3 ELISA Bmax (OD405 at 10 pg / ml) Base (OD405) Bmax (OD405 at 10 pg / ml) Base (OD4Q5) lgG1v2 0.132 0.133 HuMab 002 (transient) 0.711 4,071 lgG4 0.222 0.182

HuMabs potential for P-selectin to bind to Fey receptors

IgG antibody-dependent cytotoxicity effects are mediated by Fcy receptors on effector cells. The binding of the HuMab 002 hybridoma - and transfectoma-derivative, as well as the mutated variants and control antibodies to effector cells expressing FcyR from human blood was studied through FACS analysis. Materials and methods:

Transfectants of FcyRI IIA1.6 or newly isolated effector cells were incubated with antibodies and antibody binding was detected with FITC-labeled rabbit anti-human IgG F (ab) ₂ (DAKO) or human anti-IgG F (ab) ₂ FITC-labeled rabbit (BD / Pharmingen). HuMab 002 (transient transfectoma - and / or hybridoma-derived material and mutated variants) was tested at a concentration of 1 pg / ml (transfectants IIA1.6) or 10 pg / ml (effector cells). The absence of primary antibody or human IgG4 (10 pg / ml) was used as a negative control. To detect FcyRI expression on IIA1.6 cells, FITC-labeled mouse human anti-CD64 (BD / Pharmingen) was used. In experiments using peripheral blood mononuclear cells enriched with NK cells, NK cells were identified by double staining using PE-labeled mouse human antiCD56 (BD / Pharmingen). Granulocytes and monocytes were identified based on the FSC / SSC profile.

IIA1.6 cells, IIA1.6-Fc yRI transfectants and newly isolated effector cells were incubated with antibodies. Antibody binding was detected with F (ab) ₂ of FITC-labeled Rb-a-hulgG (DAKO) or

.........

F (ab) ₂ of FITC-labeled Rb-a-hulgG (BD / Pharmingen).

HuMab 002 (transient transfectome, derived hybridoma- and variant material with mutation) was tested at a concentration of 1 pg / ml in the IIA1.6-FcyRl transfectant binding assay. IIA1.6 cells of the wild type were used as a negative control. As a control for FcyRI expression, m-a-huCD64-FITC (BD / Pharmingen) was used.

HuMab 002 (transient transfectome, hybridoma-derivative and mutated variant material) was tested at a concentration of 10 pg / ml in effector cell binding assays. Transient transfectoma material was not tested in the granulocyte binding assay. IgG4 (10 pg / ml) was used as a negative control in all effector cell binding assays, except for the granulocyte binding assay.

Whole blood was enriched with NK cells using an NK isolation kit (Dynal Biotech ASA, Oslo, Norway). NK cells were identified by staining with m-a-huCD56-FITC.

PBMCs (peripheral blood mononuclear cells) were obtained from whole blood using the Ficoll procedure as described in the protocol contained in the NK isolation kit (Dynal Biotech ASA, Oslo, Norway). Monocytes were identified based on the FSC / SSC profile. Granulocytes were isolated from whole blood using FACS lysis buffer and identified based on the FSC / SSC profile.

Freshly isolated effector cells were incubated with antibodies , and antibody binding was detected with F (ab ' ) ₂ rabbit anti-human IgG FITC-labeled (DAKO) or IgG F (ab) ₂ anti-IgG rabbit human FITC-labeled ( BD / Pharmingen). HuMab 002 (transient transfectoma and / or hybridoma-derived material and mutated variants) were tested at a concentration of 10 pg / ml. The absence of primary antibody or human IgG4 (10 pg / ml) was used as a negative control. NK cells were isolated from MNC samples using an NK isolation kit (Miltenyi Biotec, USA). In experiments using peripheral blood mononuclear cells enriched with NK cells, NK cells were identified by double staining using PE-labeled mouse human anti-CD56 (BD / Pharmingen). Granulocytes and monocytes were isolated according to the state of the art from PBMC (for example, Monocyte Isolation Kit (Miltenyi, see above). Granulocytes and monocytes were identified based on the FSC / SSC profile.

Results:

HuMab 002 according to the invention was able to bind to FcR, as indicated by binding to granulocytes, monocytes and NK cells. All three tested Fc-variants (lgG4v1, lgG1v1 and lgG1v2) had completely lost the ability to bind to NK cells (Table 4). In addition, HuMab 002 bound effectively to granulocytes and monocytes, while mutant variants showed levels of binding comparable to the absence of primary antibody or human IgG4, as indicated by the percentages of cells that bind to the antibody in Tables 5 and 6. This indicates that the mutant variants lose the ability to interact with the FcR on effector cells.

Since HuMab 002 can interact effectively with FcR, this antibody has the intrinsic potential to induce antibody-dependent cell-mediated cytotoxicity in vivo. Inactivation of the interaction with FcR, as performed for the Fc-variants according to the invention, effectively prevents ADCC.

TABLE 4 Antibody Binding to NK cells (% of antibodies that bind to NK cells) No antibody 0.03 HuMab 002 (hybridoma) 90.92 HuMab 002 (transient) 37.40 Human lgG4 ___________________ 0.06 ___________________ lgG4v1 0.06 lgG1v1 0.12 _________lgG1v2_________  0.00

TABLE 5 Antibody Binding to monocytes (% of antibodies that bind to monocytes) No antibody 8.5 HuMab 002 (hybridoma) 38.4 HuMab 002 (transient) 31.3 Human lgG4 9.4 lgG4v1 14.5 lgG1v1 12.3 lgG1v2 14.0

TABLE 6 Antibody Granulocyte binding (% of antibodies that bind to granulocytes) No antibody 1.2 HuMab 002 (hybridoma) 63.6 lgG4v1 1.6 lgG1v1 2.1 lgG1v2 2.0

Claims (14)

1. Antibody that binds to P-selectin, does not bind to complement factor C1q nor to Fcy receptors in NK cells, containing a Fc part derived from human origin, characterized by the fact that it is an antibody of the human IgG4 subclass in whereas S228 is replaced by P and L235 is replaced by E, by inhibiting the adhesion of leukocyte-like HL60 cells to purified P-selectin with IC50 values in the range of 0.08 to 0.5 pg / ml, and comprising CDR1, CDR2 and CDR3 regions of the light chain variable domain defined by the amino acid sequence SEQ ID NO: 3 and the CDR1, CDR2 and CDR3 regions of the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 4. 2. Antibody that binds to P-selectin, characterized by the fact that it is a human or humanized antibody. Antibody according to claim 1 or 2, characterized in that it comprises the light chain variable domain defined by the amino acid sequence SEQ ID NO: 3 and the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 4. Antibody according to any one of claims 1 to 3, characterized in that the human heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 28 and the κ-light chain constant region as defined in SEQ ID NO: 23. 5. Nucleic acid molecule, characterized by the fact that it encodes an antibody molecule, as defined in any one of claims 1 to 4. 6. Vector, characterized by the fact that it comprises the nucleic acid molecule, as defined in claim 5. 7. Method for the preparation of an antibody molecule, as defined in any one of claims 1 to 4, characterized in that it comprises cultivating a host cell comprising the vector, as defined in claim 6, under conditions that allow the synthesis of the said antibody molecule, and recovering said antibody molecule from said culture. Petition 870190057809, of 06/24/2019, p. 5/10 8. Composition, characterized by the fact that it comprises an antibody molecule, as defined in any one of claims 1 to 4. Composition according to claim 8, characterized in that it is a pharmaceutical or diagnostic composition. 10. Pharmaceutical composition, characterized in that it comprises an antibody, as defined in any one of claims 1 to 4, and at least one pharmaceutically acceptable excipient. 11. Use of an antibody, as defined in any of claims 1 to 4, characterized in that it is for use in therapy. 12. Use of an antibody, as defined in any of claims 1 to 4, characterized by the fact that it is for the preparation of a medicament for the prophylaxis and treatment of inflammatory and thrombotic disorders. Antibody according to any one of claims 1 to 4, characterized in that it is for the prophylaxis and treatment of inflammatory and thrombotic disorders, in which the disorder is an occlusive disease of peripheral arteries (PAOD) or critical limb ischemia (CLI ). 14. Kit, characterized in that it comprises an antibody molecule, as defined in any one of claims 1 to 4, a nucleic acid molecule, as defined in claim 5 or a vector, as defined in claim 6.